BAZ1B the Protean Protein.

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.

CRISPR mediated targeting of DUX4 distal regulatory element represses DUX4 target genes dysregulated in Facioscapulohumeral muscular dystrophy.

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.

Deletion of the XIST promoter from the human inactive X chromosome compromises polycomb heterochromatin maintenance.

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.

Two novel DXZ4-associated long noncoding RNAs show developmental changes in expression coincident with heterochromatin formation at the human (Homo sapiens) macrosatellite repeat.

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.

The WSTF-ISWI chromatin remodeling complex transiently associates with the human inactive X chromosome during late S-phase prior to BRCA1 and γ-H2AX.

Replicating the genome prior to each somatic cell division not only requires precise duplication of the genetic information, but also accurately reestablishing the epigenetic signatures that instruct how the genetic material is to be interpreted in the daughter cells. The mammalian inactive X chromosome (Xi), which is faithfully inherited in a silent state in each daughter cell, provides an excellent model of epigenetic regulation. While much is known about the early stages of X chromosome inactivation, much less is understood with regards to retaining the Xi chromatin through somatic cell division. Here we report that the WSTF-ISWI chromatin remodeling complex (WICH) associates with the Xi during late S-phase as the Xi DNA is replicated. Elevated levels of WICH at the Xi is restricted to late S-phase and appears before BRCA1 and γ-H2A.X. The sequential appearance of WICH and BRCA1/γ-H2A.X implicate each as performing important but distinct roles in the maturation and maintenance of heterochromatin at the Xi.

The macrosatellite DXZ4 mediates CTCF-dependent long-range intrachromosomal interactions on the human inactive X chromosome.

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.

YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas.

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.

Variation in array size, monomer composition and expression of the macrosatellite DXZ4.

Macrosatellites are some of the most polymorphic regions of the human genome, yet many remain uncharacterized despite the association of some arrays with disease susceptibility. This study sought to explore the polymorphic nature of the X-linked macrosatellite DXZ4. Four aspects of DXZ4 were explored in detail, including tandem repeat copy number variation, array instability, monomer sequence polymorphism and array expression. DXZ4 arrays contained between 12 and 100 3.0 kb repeat units with an average array containing 57. Monomers were confirmed to be arranged in uninterrupted tandem arrays by restriction digest analysis and extended fiber FISH, and therefore DXZ4 encompasses 36-288 kb of Xq23. Transmission of DXZ4 through three generations in three families displayed a high degree of meiotic instability (8.3%), consistent with other macrosatellite arrays, further highlighting the unstable nature of these sequences in the human genome. Subcloning and sequencing of complete DXZ4 monomers identified numerous single nucleotide polymorphisms and alleles for the three microsatellite repeats located within each monomer. Pairwise comparisons of DXZ4 monomer sequences revealed that repeat units from an array are more similar to one another than those originating from different arrays. RNA fluorescence in situ hybridization revealed significant variation in DXZ4 expression both within and between cell lines. DXZ4 transcripts could be detected originiating from both the active and inactive X chromosome. Expression levels of DXZ4 varied significantly between males, but did not relate to the size of the array, nor did inheritance of the same array result in similar expression levels. Collectively, these studies provide considerable insight into the polymorphic nature of DXZ4, further highlighting the instability and variation potential of macrosatellites in the human genome.

The Mi-2/NuRD complex associates with pericentromeric heterochromatin during S phase in rapidly proliferating lymphoid cells.

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.

The XIST noncoding RNA functions independently of BRCA1 in X inactivation.

Females with germline mutations in BRCA1 are predisposed to develop breast and ovarian cancers. A previous report indicated that BRCA1 colocalizes with and is necessary for the correct localization of XIST, a noncoding RNA that coats the inactive X chromosome (Xi) to mediate formation of facultative heterochromatin. A model emerged from this study suggesting that loss of BRCA1 in female cells could reactivate genes on the Xi through loss of the XIST RNA. However, our independent studies of BRCA1 and XIST RNA revealed little evidence to support this model. We report that BRCA1 is not enriched on XIST RNA-coated chromatin of the Xi. Neither mutation nor depletion of BRCA1 causes significant changes in XIST RNA localization or X-linked gene expression. Together, these results do not support a role for BRCA1 in promoting XIST RNA localization to the Xi or regulating XIST-dependent functions in maintaining the stability of facultative heterochromatin.

BRCA1 associates with the inactive X chromosome in late S-phase, coupled with transient H2AX phosphorylation.

The BRCA1 tumor suppressor gene encodes an E3-ubiquitin ligase that has been implicated in several distinct biochemical processes. As the cell cycle progresses, BRCA1 proteins interact transiently with nuclear foci containing DNA replication and DNA double-strand repair machinery. A hallmark of these foci is the presence of S139 phosphorylated histone H2AX. BRCA1 was recently shown to associate with facultative heterochromatin at the inactive X chromosome (Xi), where it may play a role in maintaining gene silencing. As the kinetics of this interaction has not been described, we sought to establish whether association of BRCA1 with the Xi also correlated with replication. Here we demonstrate that the interaction of BRCA1 and the Xi is transient, occurring during late S-phase. This interaction is concomitant with the presence of distinct foci of S139 phospho-H2AX and specifically corresponds with late replication of the Xi. BRCA1 and phospho-H2AX appear on the Xi immediately adjacent to CAF-1, a known marker of replication fork activity. Taken together, these data implicate BRCA1 and the H2AX kinase in replication of facultative heterochromatin on the Xi, most likely in a fashion similar to that performed at sites of DNA replication and double-strand break repair observed on somatic chromosomes.