Maintenance of Heterochromatin by the Large Subunit of the CAF-1 Replication-Coupled Histone Chaperone Requires Its Interaction with HP1a Through a Conserved Motif

Abstract

In eukaryotic cells, the organization of genomic DNA into chromatin regulates many biological processes, from the control of gene expression to the regulation of chromosome segregation. The proper maintenance of this structure upon cell division is therefore of prime importance during development for the maintenance of cell identity and genome stability. The chromatin assembly factor 1 (CAF-1) is involved in the assembly of H3-H4 histone dimers on newly synthesized DNA and in the maintenance of a higher order structure, the heterochromatin, through an interaction of its large subunit with the heterochromatin protein HP1a. We identify here a conserved domain in the large subunit of the CAF-1 complex required for its interaction with HP1a in the Drosophila fruit fly. Functional analysis reveals that this domain is dispensable for viability but participates in two processes involving heterochromatin: position-effect variegation and long range chromosomal interactions during meiotic prophase. Importantly, the identification in the large subunit of CAF-1 of a domain required for its interaction with HP1 allows the separation of its functions in heterochromatinrelated processes from its function in the assembly of H3-H4 dimers onto newly synthesized DNA. KEYWORDS heterochromatin; variegation; HP1; CAF-1; Drosophila I N eukaryotic cells, the chromatin is partitioned into two cytologically and functionally distinct structures: heterochromatin and euchromatin. Heterochromatin was initially defined as the part of the genome that remains condensed during the whole cell cycle and stains intensively with DNA dyes. Heterochromatin is generally gene poor; rich in repeated sequences and transposable elements (Hoskins et al. 2007). Initially considered to correspond to "junk DNA," heterochromatin contains essential protein-coding genes whose expression depends on the neighboring heterochromatin structure (Schulze et al. 2005). It encodes essential chromosomal structures such as centromeres (Sun et al. 1997) or telomeres (Mason et al. 2008) and is required for essential chromosomal functions such as homolog pairing during meiosis (Dernburg et al. 1996;Karpen et al. 1996) . While essential for the biology of the genome, many of these structures are not directly encoded in the sequence of these regions and epigenetic mechanisms are likely required for their maintenance through generations.The chromatin assembly factor-1 (CAF-1) is a heterotrimeric complex first isolated as a histone chaperone able to deposit H3-H4 dimers onto newly synthesized DNA during replication or repair (Smith and Stillman 1989;Gaillard et al. 1996). Its large subunit interacts directly with PCNA (Shibahara and Stillman 1999;Moggs et al. 2000) and the CAF-1 complex is found in vivo at the replication foci (Krude 1995;Taddei et al. 1999). The large subunit of CAF-1 has also been associated to the maintenance of heterochromatin: it was shown to be essential for the stable inheritance of gene silencing in et al. 1997); its absence in fission yeast led ...

Emerging roles of the histone chaperone CAF-1 in cellular plasticity

“…While these phenotypes were initially attributed to CAF-1’s classical chromatin assembly function, defects in heterochromatin maintenance could not be ruled out initially. Indeed, a more recent study showed that expression of a newly identified and evolutionarily conserved HP1 interacting domain within the large CAF-1 subunit rescues the embryo-lethal null phenotype, unmasking an unprecedented role of CAF-1 during oogenesis [55]. …”

“…In summary, given the functional diversity of the CAF-1 complex and the recent characterization of additional domains within its subunits [23,55], it will be important to dissect the interdependence between nucleosome assembly and heterochromatin regulation in the context of reprogramming, transdifferentiation and pluripotency maintenance in the future. For instance, it should be informative to assess whether mutations within the newly identified HP1-interacting domain of CAF-1, discussed above, or within regions affecting binding to Kdm1a and HDACs affect the reversion of ESCs to a totipotent state, which has thus far been ascribed to CAF-1’s nucleosome assembly function [19].…”

The histone chaperone CAF-1 cooperates with the DNA methyltransferases to maintain Cd4 silencing in cytotoxic T cells

“…CAF-1 is a conserved heterotrimeric complex comprised of Chaf1a, Chaf1b, and Rbbp4 that promotes H3-H4 deposition during DNA replication in S phase (Smith and Stillman 1989;Kaufman et al 1995). The CAF-1 complex has well-established roles in the maintenance of heterochromatin and cell identity, but its function in regulating gene expression in primary somatic cells and its coordination with DNA methylation in gene silencing have been largely unexplored (Tchenio et al 2001;Krawitz et al 2002;Houlard et al 2006;Dohke et al 2008;Quivy et al 2008;Huang et al 2010;Heyd et al 2011;Cheloufi et al 2015;Ishiuchi et al 2015;Roelens et al 2017). Mechanistically, we found that the CAF-1 subunit Chaf1a associates with the histone deacetylases (HDACs) HDAC1/2 and the histone demethylase LSD1, enzymes that counteract histone marks associated with active transcription.…”

The replicative histone chaperone CAF-1 is essential for the maintenance of identity and genome integrity in adult stem cells

“…Finally, we aimed to investigate whether the well-characterized functions of Drosophila CAF1 in histone deposition and heterochromatin maintenance were involved in the maintenance of GSC identity and genome integrity. These functions notably rely on interactions with histone H3 and HP1a, respectively (Huang et al, 2010;Roelens et al, 2017;Tyler et al, 1999). Hence, we depleted H3 and HP1a using RNAi in germ cells and compared the resulting phenotypes with those obtained by depleting P180.…”

“…The primary antibodies used are as follows: mouse anti-α-spectrin 3A9 (1:500, DSHB), rat anti-Vasa (1:10, DSHB), mouse anti-fasciclin III 7G10 (1:50, DSHB), rabbit anti-cleaved caspase 3 (1:100, Cell Signaling, 9661S), rabbit anti-phospho-Smad5 Ser463/465 (referred to as pMad, 1:100, Epitomic, 2224-1, a kind gift from L. De Koning, Institut Curie, Paris, France), rabbit anti-γH2Av (1:1000, Rockland, 600-401-914), rabbit anti-P180 [1:1000, antibody raised in rabbit against a peptide corresponding to the residues 198-421 of P180 (Roelens et al, 2017)], rabbit anti-fibrillarin (1:500, Abcam), rabbit anti-I-eleORF (1:400, kindly provided by D. Finnegan, Edinburgh University, UK), rabbit anti-H3 (1:200, Abcam, 1791) and rabbit anti-aubergine (1:500, kindly provided by P. Lasko, McGill University, Montreal, Canada). Alexa Fluor 488-, 568-or 633conjugated secondary antibodies (Life Technologies and Jackson Immunoresearch) were used at a 1:500 dilution.…”