RESUMEN
Silencing most gene expression from all but one X chromosome in female mammals provides a means to overcome X-linked gene expression imbalances with males. Central to establishing gene silencing on the inactivated X chromosome are the actions of the long non-coding RNA XIST that triggers the repackaging of the chosen X into facultative heterochromatin. While understanding the mechanisms through which XIST expression is regulated and mediates its affects has been a major focus of research since its discovery, less is known about the role XIST plays in maintaining chromatin at the human inactive X chromosome (Xi). Here, we use genome engineering to delete the promoter of XIST to knockout expression from the Xi in non-cancerous diploid human somatic cells. Although some heterochromatin features exhibit limited change at the Xi, two of those assessed showed significant reductions including histone H2A monoubiquitylation at lysine 119 and histone H3 trimethylation at lysine 27, both of which are covalent histone modifications catalyzed by the polycomb repressive complexes 1 and 2 respectively. Coupled with these reductions, we observed an occasional gain of euchromatin signatures on Xp, but despite these signs of chromatin instability, we did not observe appreciable changes in the reactivation of genes from the Xi. Collectively, these data are consistent with maintenance of dosage compensation at the Xi involving multiple redundant layers of gene silencing.
Asunto(s)
Heterocromatina , Regiones Promotoras Genéticas , ARN Largo no Codificante , Inactivación del Cromosoma X , Cromosomas Humanos X/genética , Cromosomas Humanos X/metabolismo , Femenino , Silenciador del Gen , Heterocromatina/genética , Histonas/metabolismo , Humanos , Masculino , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
X chromosome structural abnormalities are relatively common in Turner syndrome patients, in particular X isochromosomes. Reports over the last five decades examining asynchronous DNA replication between the normal X and isochromosome have clearly established that the structurally abnormal chromosome is the inactive X chromosome (Xi). Here the organization of chromatin at a deleted X chromosome, an Xq isochromosome, and two isodicentric chromosomes were examined. Consistent with previous differential staining methods, at interphase, the X isochromosome and isodicentric X chromosomes frequently formed bipartite Barr bodies, observed by fluorescence microscopy using numerous independent bona fide markers of Xi heterochromatin. At metaphase, with the exception of the pseudoautosomal region and the duplicated locus of the macrosatellite DXZ4 (if present on the abnormal X chromosome based on break points), euchromatin markers were absent from the Xi, whereas histone variant macroH2A formed reproducible banded mirror-image chromosomes. Unexpectedly, the isodicentric chromosome in 46,X,idic(X)(q28) cells, which carry a near full-length q-arm-to-q-arm fused chromosome, showed at interphase very rare instances of Xi chromatin bodies that were separated by large distances in the nucleus. Further examination using immunofluorescence and FISH support the possibility that these rare cells may represent ones in which one half of the isodicentric chromosome is active and the other half is inactive.
Asunto(s)
Cromatina/genética , Aberraciones Cromosómicas , Cromosomas Humanos X , Hibridación Genética , Inactivación del Cromosoma X , Bandeo Cromosómico , Compensación de Dosificación (Genética) , Femenino , Fibroblastos , Heterocromatina/genética , Humanos , Hibridación Fluorescente in Situ , Interfase , Metafase/genéticaRESUMEN
During interphase, the inactive X chromosome (Xi) is largely transcriptionally silent and adopts an unusual 3D configuration known as the "Barr body." Despite the importance of X chromosome inactivation, little is known about this 3D conformation. We recently showed that in humans the Xi chromosome exhibits three structural features, two of which are not shared by other chromosomes. First, like the chromosomes of many species, Xi forms compartments. Second, Xi is partitioned into two huge intervals, called "superdomains," such that pairs of loci in the same superdomain tend to colocalize. The boundary between the superdomains lies near DXZ4, a macrosatellite repeat whose Xi allele extensively binds the protein CCCTC-binding factor. Third, Xi exhibits extremely large loops, up to 77 megabases long, called "superloops." DXZ4 lies at the anchor of several superloops. Here, we combine 3D mapping, microscopy, and genome editing to study the structure of Xi, focusing on the role of DXZ4 We show that superloops and superdomains are conserved across eutherian mammals. By analyzing ligation events involving three or more loci, we demonstrate that DXZ4 and other superloop anchors tend to colocate simultaneously. Finally, we show that deleting DXZ4 on Xi leads to the disappearance of superdomains and superloops, changes in compartmentalization patterns, and changes in the distribution of chromatin marks. Thus, DXZ4 is essential for proper Xi packaging.
Asunto(s)
Cromosomas Humanos X/genética , Eliminación de Gen , Genoma Humano/genética , Repeticiones de Microsatélite/genética , Inactivación del Cromosoma X , Animales , Sitios de Unión/genética , Factor de Unión a CCCTC/metabolismo , Cromatina/genética , Cromatina/metabolismo , Mapeo Cromosómico , Femenino , Humanos , Macaca mulatta , Ratones , Unión ProteicaRESUMEN
On the male X and female active X chromosome (Xa), the macrosatellite repeat (MSR) DXZ4 is packaged into constitutive heterochromatin characterized by CpG methylation and histone H3 tri-methylated at lysine-9 (H3K9me3). In contrast, DXZ4 on the female inactive X chromosome (Xi), is packaged into euchromatin, is bound by the architectural protein CCCTC-binding factor, and mediates Xi-specific long-range cis contact with similarly packaged tandem repeats on the Xi. In cancer, male DXZ4 can inappropriately revert to a Xi-like state and other MSRs have been reported to adopt alternate chromatin configurations in response to disease. Given this plasticity, we sought to identify factors that might control heterochromatin at DXZ4. In human embryonic stem cells, we found low levels of 5-hydroxymethylcytosine at DXZ4 and that this mark is lost upon differentiation as H3K9me3 is acquired. We identified two previously undescribed DXZ4 associated noncoding transcripts (DANT1 and DANT2) that are transcribed toward DXZ4 from promoters flanking the array. Each generates transcript isoforms that traverse the MSR. However, upon differentiation, enhancer of Zeste-2 silences DANT1, and DANT2 transcription terminates prior to entering DXZ4. These data support a model wherein DANT1 and/or DANT2 may function to regulate constitutive heterochromatin formation at this MSR.
Asunto(s)
ADN Satélite , Heterocromatina/genética , ARN Largo no Codificante/genética , Diferenciación Celular/genética , Línea Celular Transformada , Ensamble y Desensamble de Cromatina , Mapeo Cromosómico , Cromosomas Humanos X , Metilación de ADN , Femenino , Expresión Génica , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Masculino , Regiones Promotoras Genéticas , Reproducibilidad de los Resultados , Inactivación del Cromosoma XRESUMEN
The human genome contains numerous large tandem repeats, many of which remain poorly characterized. Here we report a novel transfer RNA (tRNA) tandem repeat on human chromosome 1q23.3 that shows extensive copy number variation with 9-43 repeat units per allele and displays evidence of meiotic and mitotic instability. Each repeat unit consists of a 7.3 kb GC-rich sequence that binds the insulator protein CTCF and bears the chromatin hallmarks of a bivalent domain in human embryonic stem cells. A tRNA containing tandem repeat composed of at least three 7.6-kb GC-rich repeat units reside within a syntenic region of mouse chromosome 1. However, DNA sequence analysis reveals that, with the exception of the tRNA genes that account for less than 6% of a repeat unit, the remaining 7.2 kb is not conserved with the notable exception of a 24 base pair sequence corresponding to the CTCF binding site, suggesting an important role for this protein at the locus.
Asunto(s)
Cromosomas Humanos Par 1 , Elementos Aisladores , Repeticiones de Minisatélite , ARN de Transferencia/genética , Proteínas Represoras/metabolismo , Alelos , Animales , Secuencia de Bases , Sitios de Unión , Factor de Unión a CCCTC , Línea Celular , Células Cultivadas , Cromatina/metabolismo , Secuencia Conservada , ADN/química , ADN/metabolismo , Variaciones en el Número de Copia de ADN , Retrovirus Endógenos , Secuencia Rica en GC , Humanos , Meiosis/genética , Ratones , Mitosis/genética , Motivos de Nucleótidos , Primates , Secuencias Repetidas TerminalesRESUMEN
Euchromatic features are largely absent from the human inactive X chromosome (Xi), with the exception of several large tandem repeats that can be detected as euchromatin bands at metaphase. Despite residing megabases apart, these tandem repeats make frequent inactive X-specific interactions. The mouse homologue has been reported for at least one of the tandem repeats, but whether the mouse Xi is also characterized by distinct bands of euchromatin remains unknown. We examined the mouse Xi for the presence of euchromatin bands by examining the pattern of histone H3 dimethylated at lysine 4 and detected two major signals. The first band resides in the subtelomeric region of band XF5 and may correspond to the pseudoautosomal region. The second band localizes to XE3 and coincides with an extensive complex repeat composed of a large tandem and inverted repeat segment as well as several large short interspersed nuclear element (SINE)-rich tandem repeats. Fluorescence in situ hybridization reveals that sequences with homology to the repeat region are scattered along the length of the Y chromosome. Immunofluorescence analysis of histone H3 trimethylated at lysine 9 on metaphase chromosomes indicates that the repeat region corresponds to a band of constitutive heterochromatin on the male X and female active X chromosomes, whereas the euchromatin signal appears to be female specific. These data suggest that the band of euchromatin observed at XE3 is unique to the mouse Xi, comparable to the chromatin arrangement of several large tandem repeats located on the human X chromosome.
Asunto(s)
Cromosomas de los Mamíferos/genética , Eucromatina/genética , Secuencias Repetidas en Tándem/genética , Inactivación del Cromosoma X/genética , Cromosoma X/genética , Animales , Femenino , Heterocromatina , Histonas/metabolismo , Humanos , Lisina/metabolismo , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Elementos de Nucleótido Esparcido Corto/genética , Especificidad de la Especie , Cromosoma Y/genéticaRESUMEN
The human X-linked macrosatellite DXZ4 is a large tandem repeat located at Xq23 that is packaged into heterochromatin on the male X chromosome and female active X chromosome and, in response to X chromosome, inactivation is organized into euchromatin bound by the insulator protein CCCTC-binding factor (CTCF) on the inactive X chromosome (Xi). The purpose served by this unusual epigenetic regulation is unclear, but suggests a Xi-specific gain of function for DXZ4. Other less extensive bands of euchromatin can be observed on the Xi, but the identity of the underlying DNA sequences is unknown. Here, we report the identification of two novel human X-linked tandem repeats, located 58 Mb proximal and 16 Mb distal to the macrosatellite DXZ4. Both tandem repeats are entirely contained within the transcriptional unit of novel spliced transcripts. Like DXZ4, the tandem repeats are packaged into Xi-specific CTCF-bound euchromatin. These sequences undergo frequent CTCF-dependent interactions with DXZ4 on the Xi, implicating DXZ4 as an epigenetically regulated Xi-specific structural element and providing the first putative functional attribute of a macrosatellite in the human genome.
Asunto(s)
Cromosomas Humanos X/genética , Proteínas Represoras/genética , Secuencias Repetidas en Tándem/genética , Sitios de Unión , Factor de Unión a CCCTC , Línea Celular Tumoral , Epigénesis Genética , Femenino , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , MasculinoRESUMEN
Enhancers are traditionally viewed as DNA sequences located some distance from a promoter that act in cis and in an orientation-independent fashion to increase utilization of specific promoters and thereby regulate gene expression. Much progress has been made over the last decade toward understanding how these distant elements interact with target promoters, but how transcription is enhanced remains an object of active inquiry. Recent reports convey the prevalence and diversity of enhancer transcription and transcripts and support both as key factors with mechanistically distinct, but not mutually exclusive roles in enhancer function. Decoupling the causes and effects of transcription on the local chromatin landscape and understanding the role of enhancer transcripts in the context of long-range interactions are challenges that require additional attention. In this review, we focus on the possible functions of enhancer transcription by highlighting several recent enhancer RNA papers and, within the context of other enhancer studies, speculate on the role of enhancer transcription in regulating differential gene expression.
Asunto(s)
Cromatina/genética , ADN/genética , Elementos de Facilitación Genéticos/genética , ARN no Traducido/genética , Regulación de la Expresión Génica , Histonas/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
DXZ4 is an X-linked macrosatellite composed of 12-100 tandemly arranged 3-kb repeat units. In females, it adopts opposite chromatin arrangements at the two alleles in response to X-chromosome inactivation. In males and on the active X chromosome, it is packaged into heterochromatin, but on the inactive X chromosome (Xi), it adopts a euchromatic conformation bound by CTCF. Here we report that the ubiquitous transcription factor YY1 associates with the euchromatic form of DXZ4 on the Xi. The binding of YY1 close to CTCF is reminiscent of that at other epigenetically regulated sequences, including sites of genomic imprinting, and at the X-inactivation centre, suggesting a common mode of action in this arrangement. As with CTCF, binding of YY1 to DXZ4 in vitro is not blocked by CpG methylation, yet in vivo both proteins are restricted to the hypomethylated form. In several male carcinoma cell lines, DXZ4 can adopt a Xi-like conformation in response to cellular transformation, characterized by CpG hypomethylation and binding of YY1 and CTCF. Analysis of a male melanoma cell line and normal skin cells from the same individual confirmed that a transition in chromatin state occurred in response to transformation.
Asunto(s)
Carcinoma/genética , Cromosomas Humanos X/metabolismo , Proteínas Represoras/metabolismo , Secuencias Repetidas en Tándem , Factor de Transcripción YY1/metabolismo , Secuencia de Bases , Factor de Unión a CCCTC , Línea Celular Tumoral , Transformación Celular Neoplásica , Células Cultivadas , Cromatina/metabolismo , Cromosomas Humanos X/química , Secuencia de Consenso , Islas de CpG , Metilación de ADN , Femenino , Histonas/metabolismo , Humanos , Masculino , Factor de Transcripción YY1/análisisRESUMEN
BACKGROUND: Williams syndrome transcription factor (WSTF) is a multifaceted protein that is involved in several nuclear processes, including replication, transcription, and the DNA damage response. WSTF participates in a chromatin-remodeling complex with the ISWI ATPase, SNF2H, and is thought to contribute to the maintenance of heterochromatin, including at the human inactive X chromosome (Xi). WSTF is encoded by BAZ1B, and is one of twenty-eight genes that are hemizygously deleted in the genetic disorder Williams-Beuren syndrome (WBS). RESULTS: To explore the function of WSTF, we performed zinc finger nuclease-assisted targeting of the BAZ1B gene and isolated several independent knockout clones in human cells. Our results show that, while heterochromatin at the Xi is unaltered, new inappropriate areas of heterochromatin spontaneously form and resolve throughout the nucleus, appearing as large DAPI-dense staining blocks, defined by histone H3 lysine-9 trimethylation and association of the proteins heterochromatin protein 1 and structural maintenance of chromosomes flexible hinge domain containing 1. In three independent mutants, the expression of a large number of genes were impacted, both up and down, by WSTF loss. CONCLUSIONS: Given the inappropriate appearance of regions of heterochromatin in BAZ1B knockout cells, it is evident that WSTF performs a critical role in maintaining chromatin and transcriptional states, a property that is likely compromised by WSTF haploinsufficiency in WBS patients.
Asunto(s)
Heterocromatina/metabolismo , Síndrome de Williams/genética , Secuencia de Bases , Línea Celular , Ensamble y Desensamble de Cromatina , Cromosomas Humanos X , Técnicas de Inactivación de Genes , Haploinsuficiencia/genética , Histonas/genética , Histonas/metabolismo , Humanos , Regiones Promotoras Genéticas , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating muscle disease that currently does not have an effective cure or therapy. The abnormal reactivation of DUX4, an embryonic gene that is epigenetically silenced in somatic tissues, is causal to FSHD. Disease-specific reactivation of DUX4 has two common characteristics, the presence of a non-canonical polyadenylation sequence within exon 3 of DUX4 that stabilizes pathogenic transcripts, and the loss of repressive chromatin modifications at D4Z4, the macrosatellite repeat which encodes DUX4. We used CRISPR/Cas9 to silence DUX4 using two independent approaches. We deleted the DUX4 pathogenic polyadenylation signal, which resulted in downregulation of pathogenic DUX4-fl transcripts. In another approach, we transcriptionally repressed DUX4 by seeding heterochromatin using the dCas9-KRAB platform within exon 3. These feasibility of targeting DUX4 experiments were initially tested in a non-myogenic carcinoma cell line that we have previously characterized. Subsequently, in an immortalized patient myoblast cell line, we demonstrated that targeting DUX4 by either approach led to substantial downregulation of not only pathogenic DUX4 transcripts, but also a subset of its target genes that are known biomarkers of FSHD. These findings offer proof-of-concept of the effect of silencing the polyadenylation sequence on pathogenic DUX4 expression.
Asunto(s)
Proteínas de Homeodominio/genética , Distrofia Muscular Facioescapulohumeral/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transcripción Genética , Adulto , Sistemas CRISPR-Cas/genética , Epigenómica , Femenino , Marcación de Gen , Células HCT116 , Heterocromatina/genética , Humanos , Masculino , Células Musculares/metabolismo , Distrofia Muscular Facioescapulohumeral/patología , Cultivo Primario de Células , ARN MensajeroRESUMEN
The bromodomain adjacent to the zinc finger domain 1B (BAZ1B) or Williams syndrome transcription factor (WSTF) are just two of the names referring the same protein that is encoded by the WBSCR9 gene and is among the 26-28 genes that are lost from one copy of 7q11.23 in Williams syndrome (WS: OMIM 194050). Patients afflicted by this contiguous gene deletion disorder present with a range of symptoms including cardiovascular complications, developmental defects as well as a characteristic cognitive and behavioral profile. Studies in patients with atypical deletions and mouse models support BAZ1B hemizygosity as a contributing factor to some of the phenotypes. Focused analysis on BAZ1B has revealed this to be a versatile nuclear protein with a central role in chromatin remodeling through two distinct complexes as well as being involved in the replication and repair of DNA, transcriptional processes involving RNA Polymerases I, II, and III as well as possessing kinase activity. Here, we provide a comprehensive review to summarize the many aspects of BAZ1B function including its recent link to cancer.
Asunto(s)
Factores de Transcripción/genética , Síndrome de Williams/genética , Animales , Ensamble y Desensamble de Cromatina , Reparación del ADN , Humanos , Neurogénesis , Factores de Transcripción/metabolismo , Síndrome de Williams/metabolismoRESUMEN
BACKGROUND: Macrosatellites are some of the largest variable number tandem repeats in the human genome, but what role these unusual sequences perform is unknown. Their importance to human health is clearly demonstrated by the 4q35 macrosatellite D4Z4 that is associated with the onset of the muscle degenerative disease facioscapulohumeral muscular dystrophy. Nevertheless, many other macrosatellite arrays in the human genome remain poorly characterized. RESULTS: Here we describe the organization, tandem repeat copy number variation, transmission stability and expression of four macrosatellite arrays in the human genome: the TAF11-Like array located on chromosomes 5p15.1, the SST1 arrays on 4q28.3 and 19q13.12, the PRR20 array located on chromosome 13q21.1, and the ZAV array at 9q32. All are polymorphic macrosatellite arrays that at least for TAF11-Like and SST1 show evidence of meiotic instability. With the exception of the SST1 array that is ubiquitously expressed, all are expressed at high levels in the testis and to a lesser extent in the brain. CONCLUSIONS: Our results extend the number of characterized macrosatellite arrays in the human genome and provide the foundation for formulation of hypotheses to begin assessing their functional role in the human genome.
Asunto(s)
Inestabilidad Cromosómica/genética , Variaciones en el Número de Copia de ADN/genética , Regulación de la Expresión Génica , Genoma Humano/genética , Repeticiones de Minisatélite/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Secuencia de Aminoácidos , Animales , Línea Celular , Cromosomas Humanos/genética , Secuencia Conservada/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Primates/genética , Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/química , Factor de Transcripción TFIID/genéticaRESUMEN
Almost half of the human genome consists of repetitive DNA. Understanding what role these elements have in setting up chromatin states that underlie gene and chromosome function in complex genomes is paramount. The function of some types of repetitive DNA is obvious by virtue of their location, such as the alphoid arrays that define active centromeres. However, there are many other types of repetitive DNA whose evolutionary origins and current roles in genome biology remain unknown. One type of repetitive DNA that falls into this class is the macrosatellites. The relevance of these sequences to disease is clearly demonstrated by the 4q macrosatellite (D4Z4), whereupon contraction in the size of the array is associated with the onset of facioscapulohumeral muscular dystrophy. Here, I describe recent findings relating to the chromatin organization of D4Z4 and that of the X-linked macrosatellite DXZ4, highlighting the fact that these enigmatic sequences share more than a similar name.
Asunto(s)
Epigénesis Genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Cromatina/genética , Metilación de ADN/genética , Humanos , Distrofia Muscular Facioescapulohumeral/genéticaRESUMEN
Chromosomal replication results in the duplication not only of DNA sequence but also of the patterns of histone modification, DNA methylation, and nucleoprotein structure that constitute epigenetic information. Pericentromeric heterochromatin in human cells is characterized by unique patterns of histone and DNA modification. Here, we describe association of the Mi-2/NuRD complex with specific segments of pericentromeric heterochromatin consisting of Satellite II/III DNA located on human chromosomes 1, 9, and 16 in some but not all cell types. This association is linked in part to DNA replication and chromatin assembly and may suggest a role in these processes. Mi-2/NuRD accumulation is independent of Polycomb association and is characterized by a unique pattern of histone modification. We propose that Mi-2/NuRD constitutes an enzymatic component of a pathway for assembly and maturation of chromatin utilized by rapidly proliferating lymphoid cells for replication of constitutive heterochromatin.
Asunto(s)
Autoantígenos/metabolismo , ADN Helicasas/metabolismo , Heterocromatina/metabolismo , Histona Desacetilasas/metabolismo , Linfocitos/metabolismo , Fase S , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Centrómero/genética , Centrómero/metabolismo , Técnica del Anticuerpo Fluorescente , Heterocromatina/genética , Humanos , Factor de Transcripción Ikaros/metabolismo , Inmunohistoquímica , Hibridación Fluorescente in Situ , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Microscopía Confocal , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 1 , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismoRESUMEN
Despite evidence implicating the Polycomb group protein, Eed (embryonic ectoderm development protein) in imprinted X inactivation, a similar role in random X inactivation in the embryo has remained an open question. Brockdorff and colleagues now report that Eed, along with its binding partner Enx1, transiently associates with the inactive X chromosome (Xi) and likely contributes to the epigenetic signature and long-term stability of the Xi heterochromatin.
Asunto(s)
Compensación de Dosificación (Genética) , Células Eucariotas/metabolismo , N-Metiltransferasa de Histona-Lisina , Proteínas Represoras/genética , Células Madre Totipotentes/metabolismo , Cromosoma X/genética , Animales , Regulación de la Expresión Génica/genética , Histona Metiltransferasas , Humanos , Metiltransferasas/genética , Metiltransferasas/metabolismo , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Proteína Metiltransferasas , ARN Largo no Codificante , ARN no Traducido/genéticaRESUMEN
One of several features acquired by chromatin of the inactive X chromosome (Xi) is enrichment for the core histone H2A variant macroH2A within a distinct nuclear structure referred to as a macrochromatin body (MCB). In addition to localizing to the MCB, macroH2A accumulates at a perinuclear structure centered at the centrosome. To better understand the association of macroH2A1 with the centrosome and the formation of an MCB, we investigated the distribution of macroH2A1 throughout the somatic cell cycle. Unlike Xi-specific RNA, which associates with the Xi throughout interphase, the appearance of an MCB is predominantly a feature of S phase. Although the MCB dissipates during late S phase and G2 before reforming in late G1, macroH2A1 remains associated during mitosis with specific regions of the Xi, including at the X inactivation center. This association yields a distinct macroH2A banding pattern that overlaps with the site of histone H3 lysine-4 methylation centered at the DXZ4 locus in Xq24. The centrosomal pool of macroH2A1 accumulates in the presence of an inhibitor of the 20S proteasome. Therefore, targeting of macroH2A1 to the centrosome is likely part of a degradation pathway, a mechanism common to a variety of other chromatin proteins.
Asunto(s)
Cromatina/metabolismo , Compensación de Dosificación (Genética) , Histonas/metabolismo , Animales , Ciclo Celular , Línea Celular , Núcleo Celular/metabolismo , Centrosoma/metabolismo , Mapeo Cromosómico , Femenino , Histonas/química , Histonas/genética , Humanos , Masculino , Metilación , Ratones , Fase SRESUMEN
Appreciation is growing for how chromosomes are organized in three-dimensional space at interphase. Microscopic and high throughput sequence-based studies have established that the mammalian inactive X chromosome (Xi) adopts an alternate conformation relative to the active X chromosome. The Xi is organized into several multi-megabase chromatin loops called superloops. At the base of these loops are superloop anchors, and in humans three of these anchors are composed of large tandem repeat DNA that include DXZ4, Functional Intergenic Repeating RNA Element, and Inactive-X CTCF-binding Contact Element (ICCE). Each repeat contains a high density of binding sites for the architectural organization protein CCCTC-binding factor (CTCF) which exclusively associates with the Xi allele in normal cells. Removal of DXZ4 from the Xi compromises proper folding of the chromosome. In this study, we report the characterization of the ICCE tandem repeat, for which very little is known. ICCE is embedded within an intron of the Nobody (NBDY) gene locus at Xp11.21. We find that primary DNA sequence conservation of ICCE is only retained in higher primates, but that ICCE orthologs exist beyond the primate lineage. Like DXZ4, what is conserved is organization of the underlying DNA into a large tandem repeat, physical location within the NBDY locus and conservation of short DNA sequences corresponding to specific CTCF and Yin Yang 1 binding motifs that correlate with female-specific DNA hypomethylation. Unlike DXZ4, ICCE is not common to all eutherian mammals. Analysis of certain ICCE CTCF motifs reveal striking similarity with the DXZ4 motif and support an evolutionary relationship between DXZ4 and ICCE.
Asunto(s)
Factor de Unión a CCCTC/genética , Secuencias Repetidas en Tándem , Inactivación del Cromosoma X , Animales , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Metilación de ADN , Femenino , Sitios Genéticos , Humanos , Mamíferos , Filogenia , Cromosoma X/genéticaRESUMEN
BACKGROUND: The product of dosage compensation in female mammals is the inactive X chromosome (Xi). Xi facultative heterochromatin is organized into two different types, one of which is defined by histone H3 trimethylated at lysine 9 (H3K9me3). The rationale for this study was to assess SET domain bifurcated 1 (SETDB1) as a candidate for maintaining this repressive modification at the human Xi. RESULTS: Here, we show that loss of SETDB1 does not result in large-scale H3K9me3 changes at the Xi, but unexpectedly we observed striking decompaction of the Xi territory. Close examination revealed a 0.5 Mb region of the Xi that transitioned from H3K9me3 heterochromatin to euchromatin within the 3' end of the IL1RAPL1 gene that is part of a common chromosome fragile site that is frequently deleted or rearranged in patients afflicted with intellectual disability and other neurological ailments. Centrally located within this interval is a powerful enhancer adjacent to an ERVL-MaLR element. In the absence of SETDB1, the enhancer is reactivated on the Xi coupled with bidirectional transcription from the ERVL-MaLR element. Xa deletion of the enhancer/ERVL-MaLR resulted in loss of full-length IL1RAPL1 transcript in cis, coupled with trans decompaction of the Xi chromosome territory, whereas Xi deletion increased detection of full-length IL1RAPL1 transcript in trans, but did not impact Xi compaction. CONCLUSIONS: These data support a critical role for SETDB1 in maintaining the ERVL-MaLR element and adjacent enhancer in the 3' end of the IL1RAPL1 gene in a silent state to facilitate Xi compaction.