Genetic and epigenetic characteristics of FSHD-associated 4q and 10q D4Z4 that are distinct from non-4q/10q D4Z4 homologs.

Facioscapulohumeral dystrophy (FSHD) is one of the most prevalent muscular dystrophies. The majority of FSHD cases are linked to a decreased copy number of D4Z4 macrosatellite repeats on chromosome 4q (FSHD1). Less than 5% of FSHD cases have no repeat contraction (FSHD2), most of which are associated with mutations of SMCHD1. FSHD is associated with the transcriptional derepression of DUX4 encoded within the D4Z4 repeat, and SMCHD1 contributes to its regulation. We previously found that the loss of heterochromatin mark (i.e., histone H3 lysine 9 tri-methylation (H3K9me3)) at D4Z4 is a hallmark of both FSHD1 and FSHD2. However, whether this loss contributes to DUX4 expression was unknown. Furthermore, additional D4Z4 homologs exist on multiple chromosomes, but they are largely uncharacterized and their relationship to 4q/10q D4Z4 was undetermined. We found that the suppression of H3K9me3 results in displacement of SMCHD1 at D4Z4 and increases DUX4 expression in myoblasts. The DUX4 open reading frame (ORF) is disrupted in D4Z4 homologs and their heterochromatin is unchanged in FSHD. The results indicate the significance of D4Z4 heterochromatin in DUX4 gene regulation and reveal the genetic and epigenetic distinction between 4q/10q D4Z4 and the non-4q/10q homologs, highlighting the special role of the 4q/10q D4Z4 chromatin and the DUX4 ORF in FSHD.

Cohesin mediates chromatin interactions that regulate mammalian ß-globin expression.

The ß-globin locus undergoes dynamic chromatin interaction changes in differentiating erythroid cells that are thought to be important for proper globin gene expression. However, the underlying mechanisms are unclear. The CCCTC-binding factor, CTCF, binds to the insulator elements at the 5' and 3' boundaries of the locus, but these sites were shown to be dispensable for globin gene activation. We found that, upon induction of differentiation, cohesin and the cohesin loading factor Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation. Nipbl-dependent cohesin binding is critical for long-range chromatin interactions, both between the CTCF insulator elements and between the LCR distal enhancer and the target gene. We show that the latter interaction is important for globin gene expression in vivo and in vitro. Furthermore, the results indicate that such cohesin-mediated chromatin interactions associated with gene regulation are sensitive to the partial reduction of Nipbl caused by heterozygous mutation. This provides the first direct evidence that Nipbl haploinsufficiency affects cohesin-mediated chromatin interactions and gene expression. Our results reveal that dynamic Nipbl/cohesin binding is critical for developmental chromatin organization and the gene activation function of the LCR in mammalian cells.

The effect of Nipped-B-like (Nipbl) haploinsufficiency on genome-wide cohesin binding and target gene expression: modeling Cornelia de Lange syndrome.

BACKGROUND: Cornelia de Lange syndrome (CdLS) is a multisystem developmental disorder frequently associated with heterozygous loss-of-function mutations of Nipped-B-like (NIPBL), the human homolog of Drosophila Nipped-B. NIPBL loads cohesin onto chromatin. Cohesin mediates sister chromatid cohesion important for mitosis but is also increasingly recognized as a regulator of gene expression. In CdLS patient cells and animal models, expression changes of multiple genes with little or no sister chromatid cohesion defect suggests that disruption of gene regulation underlies this disorder. However, the effect of NIPBL haploinsufficiency on cohesin binding, and how this relates to the clinical presentation of CdLS, has not been fully investigated. Nipbl haploinsufficiency causes CdLS-like phenotype in mice. We examined genome-wide cohesin binding and its relationship to gene expression using mouse embryonic fibroblasts (MEFs) from Nipbl+/- mice that recapitulate the CdLS phenotype. RESULTS: We found a global decrease in cohesin binding, including at CCCTC-binding factor (CTCF) binding sites and repeat regions. Cohesin-bound genes were found to be enriched for histone H3 lysine 4 trimethylation (H3K4me3) at their promoters; were disproportionately downregulated in Nipbl mutant MEFs; and displayed evidence of reduced promoter-enhancer interaction. The results suggest that gene activation is the primary cohesin function sensitive to Nipbl reduction. Over 50% of significantly dysregulated transcripts in mutant MEFs come from cohesin target genes, including genes involved in adipogenesis that have been implicated in contributing to the CdLS phenotype. CONCLUSIONS: Decreased cohesin binding at the gene regions is directly linked to disease-specific expression changes. Taken together, our Nipbl haploinsufficiency model allows us to analyze the dosage effect of cohesin loading on CdLS development.

Condensin I recruitment to base damage-enriched DNA lesions is modulated by PARP1.

Condensin I is important for chromosome organization and segregation in mitosis. We previously showed that condensin I also interacts with PARP1 in response to DNA damage and plays a role in single-strand break repair. However, whether condensin I physically associates with DNA damage sites and how PARP1 may contribute to this process were unclear. We found that condensin I is preferentially recruited to DNA damage sites enriched for base damage. This process is dictated by PARP1 through its interaction with the chromosome-targeting domain of the hCAP-D2 subunit of condensin I.