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Gene potentiation: Forming long-range open chromatin structures

Dr. Stephen A. Krawetz et. al.

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Gene Therapy and Molecular Biology Vol 4, page 303

Gene Ther Mol Biol Vol 4, 303-312. December 1999

Gene potentiation: Forming long-range open chromatin structures

Review Article

Susan M. Wykes1 and Stephen A. Krawetz1,2

2Department of Obstetrics and Gynecology and 1The Center for Molecular Medicine and Genetics, Wayne State University

School of Medicine, 253 C.S. Mott Center, 275 E Hancock, Detroit, MI 48201

Correspondence: Stephen A. Krawetz; Tel: (313)-577-6770; Fax: (313)-577-8554; E-mail: steve@compbio.med.wayne.edu

Key Words: chromatin domain, spermatogenesis, hematopoiesis, gene expression, chromatin structure

Received 2 August 1999; accepted 11 October 1999.


Gene potentiation is the process of opening a chromatin domain, which in turn renders genes accessible to the various factors requisite for their expression. The formation of an open chromatin structure is central to the establishment of cell fate and tissue-specific gene expression. Both hematopoiesis and spermatogenesis serve as excellent models for examining gene potentiation. Each developmental pathway is governed by a unique differentiative program, which specifies a subset of potentiated genes enabling expression. A discussion of these contrasting potentiative cascades is presented illustrating that cell fate is ultimately determined by the selective opening and closing of gene containing domains. Elucidating the mechanism, which governs these perturbations in chromatin structure, will provide valuable insight into how differentiative decisions are made and whether commitment to a particular phenotype can be modified.

I. Introduction

Many eukaryotic genes are organized into functional chromatin domains. This facilitates their coordinate regulation during development (Reviewed by Dillon and Grosveld, 1993; Vermaak et al., 1998). The ability of individual cells to regulate the genes contained within such chromatin domains is key to the establishment of cell fate and tissue-specific gene expression (Reviewed by Bonifer et al ., 1997). Perturbations in chromatin structure can act both locally to alter the accessibility of trans-acting factors to cis-regulatory elements and globally to affect the opening and closing of entire chromatin domains (Bodnar et al., 1996; Vermaak et al., 1998). Gene potentiation is the process of opening a chromatin domain (Choudhary et al.,1995), which then renders genes accessible to the various trans-acting factors required for their expression (Reviewed by Higgs, 1998). As such, transcriptionally active genes are found in regions of open chromatin.
Potentiated regions of the genome replicate early in S-phase and are preferentially confined to discreet chromosomal territories within the interphase nucleus (Kurz et al ., 1996; Lamond et al ., 1998; Wei et al.,1998). These transcriptionally competent domains exhibit a 10 fold enhanced general nuclease sensitivity (Weintraub and
Groudine, 1976). Routinely, increased DNase I sensitivity is used as a diagnostic indicator of gene potentiation. For example, the chicken ovalbumin domain is part of a multigenic, coordinately-expressed locus that exists as a single DNase I-sensitive, potentiated domain in hen oviduct where its members are expressed but remains in a DNase I-insensitive, non-potentiated configuration in all non-expressing cells (Lawson et al ., 1982). A potentiated chromatin domain may also contain small hypersensitive sequences which are approximately 100 fold more sensitive to DNase I digestion than bulk chromatin (Stalder, 1980). These hypersensitive regions often demarcate sites of interaction between specific effector proteins and cis-regulatory elements (Elgin, 1984).
The transition from a closed to an open chromatin conformation is a necessary event, but alone is not sufficient to ensure transcription (Reviewed by Krawetz et al.,1999). Consider the contrasting environments of the human -globin domain on chromosome 11 and its co-regulated family member, the -globin domain found near the telomere of chromosome 16. Both domains assume a DNase I-sensitive, potentiated conformation in erythroid cells where their respective globin genes are expressed. However, unlike the - globin domain which forms a DNase I-insensitive, closed configuration in non-erythroid cells, the -globin domain

Wykes and Krawetz: Gene potentiation in chromatin domains

remains constitutively potentiated in all cell types
(Craddock et al ., 1995). This can can be reconciles as the
-globin domain lying within a chromosomal region containing several widely expressed genes including the constitutively expressed 3-methlyadenine DNA glycosylase gene (Vyas et al ., 1992; Vickers et al ., 1993; Flint et al., 1997). Although the -globin domain remains in a potentiated conformation in non-erythroid cells, the globin genes remain transcriptionally silent because the necessary factors for its expression are lacking. It is also interesting to note that the human -globin domain exists in a potentiated, open conformation prior to commitment to the erythroid lineage (Jiménez et al., 1992) but requires additional elements and factors for appropriate expression (Calzolari et al., 1999). Taken together, these data provide supportive evidence that while the formation of a potentiated chromatin domain is a necessary event for transcription, additional levels of control are required to ensure proper spatial and temporal gene expression.
The potentiated state of a gene can also be influ- enced by alterations in the local chromatin environment. For example, many eukaryotic genes are differentially expressed by altering their methylation status. These genes are largely unmethylated in cells where they are transcribed, but fully methylated in all non-expressing cells (Reviewed by Cedar, 1988). Histone acetylation also acts on the local gene environment during the transition from the 30 nm fiber to the more open structure that can be likened to a 10 nm fiber, stabilizing the more relaxed open structure (Reviewed by Davie and Hendzel, 1994). It has also been postulated that DNA methylation patterns may serve to modulate histone acetylation thereby maintaining local chromatin states. Both DNA methylation and histone acetylation render increased accessibility of ubiquitous and tissue-specific trans-acting factors to cis-regulatory elements, facilitating transcriptional activation (Eden et al.,
In addition to enhanced general nuclease sensitivity, transcriptionally active genes are often associated with the nuclear matrix (Ciejek et al., 1983). This interaction has been postulated to represent the means by which potentiated chromatin domains are organized within the eukaryotic nucleus (Reviewed by Bode et al., 1996). Matrix attachment regions (MARs) have been shown to demarcate the boundary elements of DNase I-sensitive chromatin domains of many eukaryotic loci including the human apolipoprotein-B gene, the chicken lysozyme gene and the human PRM1 PRM2 TNP2 multigenic locus (Kalos and Fournier, 1995; Stief et al ., 1989, Kramer and Krawetz, 1996). MARs can also function as insulators against the position effects of neighboring chromatin (Zlatanova and van Holde, 1992), and have been shown to interact directly with enhancer elements to extend the accessibility of a chromatin domain (Jenuwein et al., 1997; Forrester et al., 1994). Taken together, these and other
observations have led to the suggestion that MARs can be divided into discrete functional classes and may provide a means to tag genic domains for coordinate expression (Kramer and Krawetz, 1996). While it is apparent that gene potentiation involves alterations that affect both the local environment of individual genes and the physical structure and organization of large chromatin domains containing multigene families, the actual mechanism(s) governing this process remain unclear.

II. Models for Examining Gene


A. Hematopoiesis

Hematopoiesis is the differentiative pathway by which pluripotent hematopoietic stem cells give rise to the various erythroid, lymphoid and myeloid blood cell lineages. A schematic representation of hematopoiesis is shown in Figure

1. This process initiates when a pluripotent hematopoietic

stem cell differentiates to form a myeloid or lymphoid stem cell. Both of these multipotent stem cells are capable of self renewal and differentiation. Upon appropriate stimuli, the lymphoid stem cell differentiates to form the committed pre-B and pre-T progenitor cells which in turn differentiate to form mature B and T lymphocytes respectively. In contrast, the myeloid stem cell forms an intermediate stem cell, CFU- GEMM (colony forming unit- granulocyte, erythrocyte, monocyte, megakaryocyte), which differentiates to produce the corresponding unipotent CFU-progenitors for these lineages. Subsequently, these committed precursors, influenced by various cytokines and growth factors, terminally differentiate into their respective mature erythroid, megakaryocytic, monocytic, neutrophilic, eosinophilic and basophilic cell types (Carr and Rodak, 1999).
Hematopoiesis originates in the embryonic yolk sac, temporarily shifts to the fetal liver, and from the fourteenth week of gestation throughout adult life occurs primarily in the bone marrow (Yawata, 1996). Concomitantly, many hematopoietic-specific genes are coordinately regulated and differentially expressed during development (Reviewed by Orkin, 1995). Two of the most extensively characterized examples are the human and globin gene clusters which encode the respective globin chains of hemoglobin, the oxygen transport protein found in erythrocytes (Reviewed by Karlson and Nienhuis, 1985). The human -globin gene cluster is a well-established model for examining the functional role of chromatin in gene regulation. The human - globin locus is a multigenic domain containing five globin genes arranged in the order of their sequential expression during development (Reviewed by Andrin and Spencer, 1994). The entire -globin gene cluster exists in a single DNase I- sensitive, open conformation in cells of the erythroid lineage, but remains in a DNase I-insensitive, closed conformation in all non-erythroid cells (Groudine et al ., 1983). However, the extent of the human -globin domain remains to be clearly

Gene Therapy and Molecular Biology Vol 4, page 305

Figure 1 Hematopoiesis: The differentiation and maturation of blood cells. During, hematopoiesis multipotent stem cells, influenced by numerous cytokines and growth factors, divide and differentiate to form committed progenitors for the various erythroid, lymphoid and myeloid blood cell lineages. These unipotent precursors in turn differentiate to produce their respective mature blood cell types. Each step of this differentiative pathway involves the selective restriction of chromatin domains ultimately defining a lineage-specific subset of genes which remains potentiated for expression.

delineated. Upstream from the gene cluster is a series of five DNase I-hypersensitive sites (HS1-HS5). Hypersensitive sites 1-4 are commonly referred to as the -globin locus control region, LCR (Reviewed by Grosveld et al., 1993). The importance of the -globin LCR has been widely demonstrated through the characterization of its individual hypersensitive sites (Reviewed by Dillon and Grosveld,
1993) and by examining the effects of naturally occurring
deletions which result in the various -thalassemias (Van der Ploeg et al., 1980; Driscol et al., 1989). These de-novo mutations not only lead to reduced levels of gene expression, but have also been demonstrated to alter both the timing of replication of the -globin locus and its sensitivity to DNase I (Forrester et al ., 1990). Dissection and characterization of individual hypersensitive sites has shown that the -globin LCR functions in a tissue-specific manner and is necessary for high level expression of the globin genes. Hypersensitive sites 1-4 act as erythroid-specific enhancers and possess multiple binding sites for various transcription factors (Reviewed by Engel, 1993; Wood, 1996). HS-2 has been shown to be functionally equivalent to the full LCR both by
displaying classical enhancer activity in transient transfection assays (Tuan et al., 1989) and by its ability to confer high-level, position-independent expression of a transgene in mice (Philipsen et al.,1990; Lui et al., 1992 and Pawlik et al., 1995). HS-3 possesses a dominant chromatin opening and remodeling activity that is separate and distinct from its ability to direct -globin expression in transgenic mice (Ellis et al., 1996). HS-5 is a constitutive site and although it possesses no enhancing activity (Fraser et al.,
1993), it does contain a matrix attachment region (Jarman et

al., 1988). In this manner, HS-5 may serve as an insulator against the position effects of neighboring chromatin (Li et al., 1994). These observations suggest that the LCR elements act cooperatively to ensure the correct spatial and temporal regulation of the -globin gene cluster.

The full LCR has been postulated to function synergistically as a holocomplex, to regulate the sequential expression of the -globin genes through specific HS- promoter interactions (Bresnick et al ., 1997 and Ellis et al .,
1996). While several looping models have been proposed for this developmental switching mechanism (Reviewed by

Wykes and Krawetz: Gene potentiation in chromatin domains

Stamatoyannopoulos and Nienhuis, 1994), others suggest that the temporal regulation of the -globin genes is LCR- independent, relying solely on promoter proximal elements and gene arrangement (Martin et al., 1996). Recent studies examining the organization and temporal regulation of the - globin genes have further demonstrated that inversion of gene order with respect to the LCR significantly alters their expression (Tanimoto et al., 1999).
Although it has been well established that the -globin LCR plays an essential role in the transcriptional activation and temporal regulation of the -globin locus, its function in long range chromatin opening is debated. The -globin LCR was the first element reported to confer tissue-specific, position-independent and copy number-dependent expression of a transgene (Grosveld et al., 1987). It has since been held that the -globin LCR facilitates the creation of an open chromatin environment by altering the topology of the
-globin domain thereby maintaining it in a potentiated
configuration (Reviewed by Martin et al., 1996). Conversely, others have suggested that while the LCR ensures the high-level, tissue-specific expression of the - globin genes, it does not function to form or maintain the open chromatin conformation that is necessary for the expression of the locus (Reitman et al., 1993; Reik et al.,
1998; Epner et al., 1998). This raises the intriguing possibility that the ability of the -globin LCR to overcome position effects in transgenic mice may be independent of the mechanism that governs the potentiation of the -globin domain during development (Reviewed by Higgs, 1998). Interestingly, it has been now shown that the human and mouse -globin loci reside in a cluster of functional odorant receptor genes (Bulger et al., 1999). The authors postulate that these two overlapping gene families may share some of the same regulatory elements involved in mediating their different expression patterns. Understanding how such multigenic domains are potentiated will provide valuable insight into the functional role of chromatin in the establishment of cell fate and tissue-specific gene expression.

B. Spermatogenesis

Spermatogenesis serves as another excellent model for examining gene potentiation because of the vast array of testes-specific genes that are coordinately regulated and expressed in a specific temporal manner during the formation of the male gamete (Reviewed by McCarrey,
1998). This entire differentiative pathway from the
uncommitted stem cell to the mature spermatozoa occurs within the testes. The important cellular and molecular events of spermatogenesis are summarized in Figure 2. There are three stages to mammalian spermatogenesis: mitosis, meiosis and spermiogenesis (Junqueira et al., 1986). During mitosis, primitive type A spermatogonia either actively divide, renewing themselves or differentiate to form the intermediate and type B spermatogonia. Once type A
spermatogonia differentiate to form these cell types, they are then committed to the spermatogenic pathway. Type B spermatogonia in turn differentiate to form primary spermatocytes, which subsequently enter meiosis (Reviewed by Dym, 1994). The development of meiotic spermatocytes begins with the primary or pre-leptotene spermatocyte. DNA replication occurs at this stage resulting in a genome content of 4N. After DNA synthesis, chromatin condensation is initiated, signaling the start of meiosis. In humans, approximately 22 out of the 64 days of spermatogenesis are spent in meiosis and the majority of that in prophase I (Reviewed by Willison and Ashworth, 1987). During this process, the chromosomes undergo pairing and the formation of the synaptonemal complex. This is followed by genetic recombination between the homologous pairs. Subsequent to the exchange of genetic material, the first meitotic division occurs resulting in the formation of two secondary spermatocytes. This is quickly followed by meiosis II, reduction division, producing four haploid round spermatids (Gardner and Snustad, 1984). Some of the genes expressed during meiosis include testes-specific variants of somatic cell proteins (Reviewed by Kierzenbaum, 1994). One of the best-characterized examples is Pgk2, phosphoglycerate kinase 2, and the testes specific isozyme of Pgk1. Pgk1 is a constitutively expressed, X-linked enzyme that catalyzes the conversion of 1,3 diphosphoglycerate to 3-phosphoglycerate and ATP during glycolysis (Lee et al ., 1972). However, because it is subject to X-inactivation (Lifschytz and Lyndsay, 1972), the expression of the autosomal, testes-specific Pgk2 gene is initiated to compensate for the reduced levels of this essential enzyme during spermatogenesis (McCarrey et al.,
The final stage of spermatogenesis, termed spermiogenesis, is characterized by the morphological differentiation of round spermatids into mature spermatozoa (Clermont and Leblond, 1955). In humans, like other mammals, the round spermatid stage marks the initial expression of the haploid-specific packaging proteins the transition proteins and the protamines (Wykes et al., 1995). These proteins facilitate the remodeling of the chromatin during the morphologic transformation from round spermatid to mature spermatozoa (Reviewed by Wouters- Tyrou, 1998). This process involves the initial disruption of the nucleohistone structure by the transition proteins and their final replacement by the protamines to compact and condense the DNA into a species-specific shaped nucleus (Reviewed by Dadoune, 1995). Subsequent to their expression these genes are again suppressed as part of the genome-wide silencing that yields the transcriptionally quiescent sperm nucleus (Reviewed by Balhorn, 1989; Oliva and Dixon, 1991). Accordingly, many testes-specific genes, including the transition proteins and the protamines, are synthesized relatively early then placed under extensive translational control (Reviewed by Eddy and O’Brien,

Gene Therapy and Molecular Biology Vol 4, page 307

Figure 2 The three stages of human spermatogenesis. Human spermatogenesis is divided into three stages, mitosis, meiosis and spermiogenesis. Some of the important cellular and molecular events are indicated. During mitosis primitive type A spermatogonia either actively divide renewing themselves or differentiate to form type B spermatogonia and thus commit themselves to the spermatogenic pathway. Type B spermatogonia in turn differentiate to form primary or preleptotene spermatocytes which then enter meiosis. Meiosis initiates after DNA replication and is characterized by chromosomal pairing and genetic recombination. Subsequent to the exchange of genetic material, meiotic spermatocytes undergo two successive cell divisions producing four round haploid spermatids. During the final stage, spermiogenesis, these round spermatids then morphologically differentiate to form mature spermatozoa. This process is facilitated by the transition proteins and the protamines which mediate the remodeling of the chromatin during the terminal differentiation of the spermatid nucleus.

Spermatogenesis is a dynamic continuum reflecting the coordinate temporal regulation of genes involved in the formation of functional male gametes. It is an exquisite model for examining the mechanism of gene potentiation.

III. Selective Potentiation of Testes- Specific Domains

It is well established that the DNA in human sperm chromatin is partitioned into both a nucleohistone and nucleoprotamine fraction (Tanphaichutr et al., 1978). This organization is likely to be sequence specific and may serve to designate a particular subset of genes which have a functional role in early development (Gatewood et al.,
1987). In humans, 85% of the DNA in sperm chromatin is protamine bound, while 15% remains histone bound (Tanphaichutr et al., 1978). The histone bound fraction contains several testes-specific histone variants (Reviewed
by Ward, 1994; Donecke et al ., 1997). This compacts the DNA to a greater extent than its somatic counterparts although histone H1 is absent and histones H3 and H4 are highly acetylated (Gatewood et al., 1990). Since both histone acetylation and the absence of histone H1 are features of active chromatin (Reviewed by Wolffe, 1994), it has been proposed that the histone-associated genes in
the human sperm nucleus may be the first genes transcribed after fertilization (Gardiner-Garden et al., 1998). Alternatively, these histone bound regions may simply serve as nucleation sites for the replacement of the protamines by the histones upon fertilization for the resumption of the somatic nucleosomal structure.
The transition proteins and the protamines function solely to remodel the chromatin during late spermatogenesis. The PRM1 PRM2 TNP2 members of this sperm gene packaging family are clustered into a single 28 kb DNase I- sensitive domain flanked by two regions of marked insensitivity (Choudhary et al., 1995). A diagrammatic representation of this domain is shown in Figure 3). This biophysical domain has been biologically confirmed. Transgenic mice harboring the human protamine locus express the transgene in a haploid-specific, position-independent and copy-number dependent manner (Choudhary et al., 1995; Stewart et al ., 1999). These data show that this region of the genome contains all of the elements necessary for the appropriate temporal and spatial expression of this suite of genes independent of site of integration.
The ends of the domain are attached to the sperm nuclear matrix (Kramer and Krawetz, 1996). The sequences of attachment at the boundaries of this domain correspond to sperm-specific matrix attachment regions, termed spMARs.

Wykes and Krawetz: Gene potentiation in chromatin domains

Figure 3: The human PRM1 PRM2 TNP2 domain. The human protamine locus is a multigenic domain containing three haploid expressed genes encoding the protamines PRM1, PRM2 and transition protein TNP2. This gene cluster exists as a single DNase I-sensitive domain flanked by two regions of matrix attachment. The relative position of the locus along human chromosome 16p13.13 is shown.

Figure 4: Gene potentiation during mammalian spermatogenesis. The potentiative state of individual genic domains was assessed by DNase I sensitivity in various isolated germ cell populations ranging from Type A spermatogonia to round spermatid. The alterations in the potentiative states of Pgk1 and the testes-specific Pgk2 and Prm1 Prm2 Tnp2 domains indicates that the temporal regulation of these genes for expression during spermatogenesis is mediated by the selective opening and closing of individual chromatin domains. The constitutively expressed -actin and erythroid-specific -globin genes provide positive and negative controls respectively.


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Although the functional role of the spMAR elements in gene potentiation remains to be clearly delineated recent evidence suggests that the 3’ spMAR of the PRM1 PRM2 TNP2 domain is critical for expression of this human locus (Kramer et al., 1997).
The protamine gene cluster remains transcriptionally quiescent until the round spermatid stage when it is then actively transcribed in the final burst of transcription before genome-wide silencing (Fig. 2, Wykes et al., 1995). This is representative of the class of similarly expressed genes that constitute the alternative male haploid genome (Kramer and Krawetz, 1997). It has thus been argued that expression of this alternative genome is controlled by sequestering its members in a closed chromatin conformation until required (Kramer et al., 1998). To directly address this tenet, the potentiative state of the Prm1 Prm2 Tnp2 gene cluster and several other gene loci including Pgk1, Pgk2, -actin and -globin, was assessed throughout spermatogenesis using purified populations of germ cells (Kramer et al., 1998). The results of this study are summarized in Figure 4. It was reasoned that since, Pgk1 is a constitutively expressed gene its domain should exist in a potentiated conformation in all cell types. However, Pgk1 is also subject to X-inactivation during spermatogenesis, which is compensated by the expression of the autosomal testes-specific Pgk2. As the Pgk1 gene is silenced and its chromatin domain is closed, the closed Pgk2 domain opens to enable its transcription through to the round spermatid stage. Similarly, the multigenic Prm1 Prm2 Tnp2 domain remains closed until the pachytene spermatocyte stage when it then assumes an open potentiated chromatin conformation prior to the expression of its members in round spermatids. This demonstrated that the temporal expression of testes- specific genes is ultimately mediated by the selective opening and closing of individual chromatin domains. Interestingly, the physical domain containing the human PRM1 PRM2 TNP2 multigenic locus remains in an open conformation in mature spermatozoa (Choudhary et al., 1995).

IV. Prospects

Both hematopoiesis and spermatogenesis serve as excellent model systems for examining gene potentiation. Each developmental process begins with a progenitor stem cell, which, in response to various stimuli, acquires the capacity to differentiate into individual cell types. Commitment to a particular cell fate is a gradual process and is ultimately determined by the selective potentiation of certain genes for expression in conjunction with the repression or silencing of other genes. For example, commitment to a specific hematopoietic pathway involves the selective repression of genes to restrict lineage potential from its multipotent stem cell (Hu et al., 1997). This repressive mechanism is marked by the successive
closing of chromatin domains eventually defining a lineage- specific subset of genes that remains potentiated for expression (Jimenez et al, 1992). Conversely, spermatogenic differentiation is governed by an expressive mechanism whereby the activation of testes-specific genes is mediated by the selective opening, i.e. potentiation, of individual chromatin domains (Kramer et al., 1998). Further characterization of these contrasting potentiative mechanisms which establish cell fate during hematopoiesis and spermatogenesis will provide insight into how differentiation is regulated by chromatin structure and whether commitment to a particular cell fate can be altered (Krawetz et al., 1999). The identification of potentiator elements, which facilitate the opening and closing of chromatin domains may be useful to ensure high-level, tissue-specific expression of targeted gene therapeutics.


This work was supported by NIH grant HD36512 to S.A.K. PreDoctoral fellowship support to S.M.W. from the WSU CMMG is gratefully acknowledged.


Andrin C, and Spencer C (1994) The intricacies of -globin gene expression. Biochem. Cell Biol. 72, 377-380.

Balhorn R (1989) Mammalian protamines: structure and molecular interactions. In: Molecular Biology of Chromosome Function. Aldolph, K. (Editor) Springer-Verlag. Pp 366-395.

Bode J, Stengert-Iber M, Kay V, Schlake T, and Dietz-Pfeilsteller A (1996) Scaffold/Matrix-attached regions: Topological switches with multiple regulatory functions. Crit Rev Euk Gene Express 6, 115-138.

Bodnar J, and Bradley M (1996) A chromatin switch. J. Theor. Biol.

183, 1-7.

Bonifer C, Jägle U, and Huber M ( 1997) The chicken lysozyme locus as a paradigm for the complex developmental regulation of eukaryotic gene loci. J. Biol. Chem. 272, 26057-26078.

Bresnick E, and Tze L (1997) Synergism between hypersensitive sites confers long-range gene activation by the beta-globin locus control region. Proc Natl Acad Sci USA 94, 4566-4571.

Bulger M, von Doornick J, Saitoh N, Telling A, Farrell C, Bender M, Felsenfeld G, Axel R, and Groudine M (1999) Conservation of sequence and structure falnking the mouse and human -globin loci: The -globin genes are embedded within an array of odorant receptor genes. Proc Natl Acad Sci USA 96, 5129-


Calzolari R, McMorrow T, Yannoutsos N, Langeveld A, and Grosveld F (1999) Deletion of a region that is a candidate for the difference between the deletion forms of hereditary persistence of fetal hemoglobin and - Thalassemia affects but not globin gene expression. EMBO J. 18, 949-958.

Carr J, and Rodak B (Editors) Clinical Hematology Atlas (1999) W.B. Saunders, Philadelphia, PA, Pp 10-12.

Cedar H (1988) DNA methylation and gene activity. Cell 53, 3-4. Choudhary S, Wykes S, Kramer J, Mohammed A, Koppitch F,

Nelson J, and Krawetz S.A. (1995) A haploid expressed gene


Wykes and Krawetz: Gene potentiation in chromatin domains

cluster exists as a single chromatin domain in human sperm. J. Biol. Chem. 270, 8766-8762.

Ciejek E, Tsai M, and O’Malley B (1983) Actively transcribed genes are associated with the nuclear matrix. Nature 306,


Clermont Y, and Leblond C (1955) Spermiogenesis of man, monkey, ram and other mammals as shown by the “Periodic Acid-Schiff” Technique. Am.J. Anatomy 96,


Craddock C, Vyas P, Sharpe J, Ayyub H, Wood W, and Higgs D (1995) Contrasting effects of and globin regulatory elements on chromatin structure may be related to their different chromatin environments EMBO J. 14, 1718-


Dadoune J (1995) The nuclear status of human sperm cells.

Micron. 26, 323-345.

Davie J, and Hendzel M (1994) Multiple functions of dynamic histone acetylation. J.Cell Biochem. 55, 98-105.

Dillon N, and Grosveld F (1993) Transcriptional regulation of multigene loci: multilevel control. Trends Genet 9, 134-


Doenecke D, Drabent B, Bode C, Bramlage B, Franke K, Gavenis K, Kosciessa U, and Witt O (1997) Histone gene expression and chromatin structure during spermatogenesis. Adv. Exp. Med. Biol. 424, 37-48.

Driscoll M, Dobkin C, and Alter B (1989) - Thalassemia due to a de novo mutation deleting the 5’ -globin gene activation region hypersensitive sites. Proc Natl Acad Sci USA 86, 7470-7474.

Dym M ( 1994) Spermatogonial stem cell of the testes. Proc Natl

Acad Sci USA 91, 11287-11289.

Eddy E and O’Brien D (1998) Gene expression during mammalian meiosis. Curr. Topics Dev. Biol. 37, 141-200.

Eden S, Hashimshony T, Keshet J, Cedar H, and Thorne A (1998) DNA methylation models histone acetylation. Nature 394, 842.

Engel J (1993) Developmental regulation of human -globin gene transcription: a switch of loyalties. Trends Genet 9,


Elgin S (1984) Anatomy of hypersensitive sites. Nature 309,


Ellis J, Tan-Un K, Harper A, Michalovich D, Yannoutsos N, Philipsen S, and Grosveld F (1996) A dominant chromatin opening activity in 5’ hypersensitive site 3 of the human - globin locus control region. EMBO J. 15, 562-568.

Epner E, Reik A, Cimbora D, Telling A, Bender M, Fiering D, Enver T, Martin D, Kennedy M, Keller G, and Groudine M (1998). The -globin LCR is not necessary for an open chromatin structure or transcription of the mouse -globin locus. Mol.Cell 2, 447-455.

Flint J, Thomas K, Micklem G, Raynham H, Clark K, Doggett N, King A, and Higgs D (1997) The relationship between chromosome structure and function at the human telomeric region. Nature Genetics 15, 252-257.

Forrester W, Epner E, Driscoll M, Enver T, Brice M, Papayannopoulou T, and Groudine M (1990) A deletion of the human -globin locus activation region causes a major

alteration in chromatin structure and replication across the entire -globin locus. Genes Dev. 4, 1637-1649.

Forrester W, van Genderen C, Jenuwein T, and Grosschedl R (1994) Dependence of enhancer-mediated transcription of the immunoglobulin gene on nuclear matrix attachment regions. Science 265, 1221-1225.

Fraser P, Pruzina S, Antoniou M, and Grosveld F (1993) Each hypersensitive site of the human -globin locus control region confers a different developmental pattern of expression on the globin genes. Genes Dev. 7, 106-113.

Gardiner-Garden M, Ballesteros M, Gordon M and Tam P (1998) Histone and protamine DNA association: conservation of different patterns within the -globin domains in human sperm. Mol.Cell Biol. 18, 3350-3356.

Gardner E, and Snustad D (Editors): Principles of Genetics . Seventh

Edition. (1984) John Wiley & Sons. Pp 46-52.

Gatewood J, Cook G, Balhorn R, Bradbury E, and Schmid C (1987) Sequence-specific packaging of DNA in human sperm chromatin. Science 236, 962-964.

Gatewood J, Cook G, Balhorn R, Schmid C, and Bradbury E (1990) Isolation of four core histones from human sperm chromatin representing a minor subset of somatic histones. J Biol.Chem .

265, 20662-20666.

Grosveld F, Blom van Assendelft G, Greaves D, and Kollias G (1987) Position-independent high-level expression of the human -globin gene in transgenic mice. Cell 51, 975-985.

Grosveld F, Dillon N, and Higgs D (1993) The haemoglobinopathies.

In: Baillièris Clinical Haematology. Higgs, D. and Weathall, D. (Editors) Baillière Tindall. Pp 31-56.

Groudine M, Kohwi-Shigetmatsu T, Gelinas R, Stamatoyannopoulas G, and Papayannopoulou T (1983) Human fetal to adult globin switching: changes in chromatin structure of the -globin gene locus. Proc Natl Acad Sci USA USA 80, 7551-7555.

Higgs D (1998) Do LCRs open chromatin domains. Cell 95, 299-


Hu M, Krause K, Greaves M, Sharkis S, Dexter M, Heyworth C, and Enver T (1997) Multilineage gene expression precedes commitment in the hematopoietic system. Genes Dev. 11, 774-


Jarman A, and Higgs D (1988) Nuclear Scaffold attachment sites in the human globin gene complexes. EMBO J. 7, 3337-3344.

Jenuwein T, Forrester W, Fernandez-Herrero L, Laible G, Dull M, and Grosschedl R (1997). Extension of chromatin accessibility by nuclear matrix attachment regions. Nature 385, 269-272.

Jiménez G, Griffithe S, Ford A, Greaves M, and Enver T (1992) Activation of the -globin locus control region precedes commitment to the erythroid lineage. Proc Natl Acad Sci USA

89, 10618-10622.

Junqueira L, Carneiro J, and Long J (Editors): Basic Histology. Fifth

Edition.(1986) Appleton-Century-Crofts. Pp 468-484.

Kalos M, and Fournier R (1995) Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain. Mol. Cell. Biol. 15, 198-


Karlson S and Nienhuis A (1985) Developmental regulation of the human globin genes. Ann. Rev. Biochem. 54, 1071-1108.


Gene Therapy and Molecular Biology Vol 4, page 311

Kierszenbaum A (1994) Mammalianm spermatogenesis in vivo and in vitro: a partnership of spermatogenic and somatic cell lineages. Endoc. Rev. 15, 116-134.

Kramer J, and Krawetz, S. A. (1996) Nuclear matrix interactions within the sperm genome. J. Biol. Chem. 271, 11619-


Kramer, J, and Krawetz, S.A. ( 1997) RNA in sperm: implications for the alternative haploid genome. Molecular Human Reproduction 3:473-478.

Kramer J, Zhang S, Yaron Y, Zhao Y, and Krawetz, S. A. ( 1997) Genetic testing for male infertility: a postulated role for mutations in sperm nuclear matrix attachment regions. Genetic Testing 1, 125-129.

Kramer J, McCarrey J, Djakiew D, and Krawetz S.A. (1998) Differentiation: the selective potentiation of chromatin domains. Development 125, 4749-4755.

Krawetz S.A., Kramer J, and McCarrey J (1999) Reprogramming the male gamete genome: a window to successful gene therapy. Gene 234, 1-9.

Kurz A, Lampel S, Nickolenko J, Brandl J, Benner A, Zirbel R, Cremer T, and Lichter P (1996) Active and inactive genes localize preferentially in the periphery of chromosome territories. J. Cell Biol. 135, 1195-1205.

Lamond A, and Earnshaw (1998) Structure and function in the nucleus. Science 280, 547-553.

Lawson G, Knoll B, March C, Woo S, Tsai M, and O’Malley B (1982) Definition of 5’ and 3’ structural boundaries of the ovalbumin multigene family. J. Biol. Chem. 257, 1501-


Lee C, Niesel D, Pegoraro B, and Erikson R (1980) Immunological and structural relatedness of isozymes and genetic variants of 3-phoshoglycerate kinase from the mouse. J. Biol. Chem. 255, 2590-2595.

Li Q, and Stamatoyannopoulos G (1994) Hypersensitive-site 5 of the human -locus control region functions as a chromatin insulator. Blood 84, 1399-1401.

Lifschytz E and Lindsley D (1972). The role of X-chromosome inactivation during spermatogenesis. Proc Natl Acad Sci USA 69, 182-186.

Liu D, Chang J, Moi P, Liu W, Kan Y and Curtin P (1992) Dissection of the enhancer activity of the -globin 5’ hypersensitive site 2 in transgenic mice. Proc Natl Acad Sci USA 89, 3899-3903.

Martin D, Fiering S, and Groudine M (1996) Regulation of - globin gene expression: straightening out the locus. Curr.Opin. Gen. Dev. 6, 488-495.

McCarrey J, Berg W, Paragioudakis S, Zhang P, Dilworth D, Arnold B, and Rossi J (1992) Differential transcription of Pgk genes during spermatogenesis in the mouse. Dev. Biol.

154, 160-168.

McCarrey J, (1998) Spermatogenesis as a model system for developmental analysis of regulatory mechanisms associated with tissue-specific gene expression. Seminars Cell Dev. Biol. 9, 459-466.

Oliva R, and Dixon G ( 1991) Vertebrate protamine genes and the histone to protamine replacement reaction. In: Progress in Nucleic Acid Research and Molecular Biology. Cohn, W. and Moldave, K. (Editors) Academic Press. Pp 26-96.

Orkin S ( 1995) Transcription factors and hematopoetic development.

J. Biol. Chem. 270, 4955-4958.

Pawlik K, and Townes T (1995) Automonous, erythroid-specific DNase I hypersensitive site formed by the human -globin locus control region, LCR 5’HS2 in transgenic mice. Dev. Biol.

169, 728-732.

Philipsen S, Talbot D, Fraser P, and Grosveld F (1990) The -globin dominant control region: Hypersensitive site 2. EMBO J. 9,


Reik A, Telling A, Zitnik G, Cimbora D, Epner E, and Groudine M (1998) The locus control region is necessary for gene expression in the human -globin locus but not the maintenance of an open chromatin structure in erythroid cells. Mol. Cell. Biol. 18, 5992-6000.

Reitman M, Lee E, Westphal H, and Felsenfeld G (1993) An enhancer/locus control region is not sufficient to open chromatin. Mol. Cell. Biol. 13, 3990-3998.

Stalder J, Larsen A, Engel J, Dolan,M, Groudine M, and Weintraub H (1980) Tissue-specific DNA cleavages in the globin chromatin domain introduced by DNase I. Cell 20, 451-460.

Stamatoyannopoulos G, and Nienhuis A (1994) Hemoglobin switching. In: The Molecular Basis of Blood Diseases. Stamatoyannopoulos, G. (Editor). Philadelphia. WB Saunders. Pp 107-156.

Stewart K, Kramer J, Evans, M, and Krawetz S.A. (1999) Temporal expression of the transgenic human protamine gene cluster. Fertility and Sterility 71, 739-745.

Stief A, Winter D, Str�tling W, and Sippel A (1989) A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature 341,343-345

Tanimoto K, Liu Q, Bungert J, and Engel J (1991) Effects of altered gene order or orientation of the locus control region on human

-globin gene expression in mice. Nature 398, 344-347.

Tanphaichitr N, Sobhon P, Taluppeth N, and Chalermisarachai P (1978) Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man. Exp. Cell Res. 117, 347-356.

Tuan D, Solomon W, London I, and Lee D (1989) An erythroid- specific developmental stage-independent enhancer far upstream of the human “ -like globin” genes. Proc Natl Acad Sci USA 86, 2554-2558.

Van der Ploeg L, Konings A, Oort M, Roos D, Bernini L, and Flavell R (1980) - Thalassemia studies showing that deletion of the and genes influence -globin gene expression in man. Nature

283, 637-642.

Vermaak D, and Wolffe A (1998) Chromatin and chromosomal controls in development. Dev. Genetics 22, 1-6.

Vickers M, Vyas P, Harris P, Simmons D, and Higgs D (1993) Structure of the human 3-methyladenine DNA glycosylase gene and localization close to the 16p telomere. Proc Natl Acad Sci USA 90, 3437-3441.

Vyas P, Vickers M, Picketts D and Higgs D (1995) Conservation of position and sequence of a novel widely expressed gene containing the major human alpha-globin regulatory element. Genomics 29, 679-689.

Ward S ( 1994) The structure of the sleeping genome: implications of sperm DNA organization for somatic cells. J. Cell Biochem.

55, 77-82.


Wykes and Krawetz: Gene potentiation in chromatin domains

Wei X, Samarabandu J, Devdhar R, Siegel A, Acharya R, and Berezney R (1998) Segregation of transcription and replication sites into higher order domains. Science 281,


Weintraub H, and Groudine M (1976) Chromosomal subunits in active genes have an altered conformation. Science 193,


Willison K, and Ashworth A (1987) Mammalian spermatogenic gene expression. Trends Genet 3, 351-355.

Wolffe A (1994) Inheritance of chromatin states. Dev. Genet. 15,


Wood W (1996) The complexities of -globin gene regulation.

Trends Genet 12, 204-207.

Wouters-Tyrou D, Martinage A, Chevaillier P, and Sautiere P (1998) Nuclear basic proteins in spermiogenesis. Biochimie 80, 117-28.

Wykes S, Nelson J, Visscher D, Djakiew D, and Krawetz S.A. (1995) Coordinate expression of the PRM1, PRM2 and TNP2 multigene locus in human testes. DNA Cell Biol. 14:


Yawata Y (Editor) (1996) Atlas of Blood Diseases: Cytology and histology Martin Dunitz Ltd. London. Pp 3-6.

Zlantanova J, and van Holde K (1992) Chromatin loops and transcriptional regulation. Crit Rev Euk Gene Express 3,


Stephen A. Krawetz