Fernando Azorín

Phosphorylation is crucial for the regulation of Barrier-to-Autointegration Factor (BAF), a conserved nuclear envelope (NE)-associated protein that, among its diverse functions, binds chromatin and mediates its anchoring to the NE. In interphase, cycles of BAF phosphorylation, which inhibits chromatin binding, and dephosphorylation, which restores it, are essential for the dynamic anchoring of chromatin to the NE. During mitosis, BAF becomes highly phosphorylated and dissociates from chromatin, except for a small centromeric pool (cenBAF). Upon mitotic exit, BAF is dephosphorylated and re-associates with chromosomes. Two Ser/Thr protein phosphatases, PP2A and PP4, are known to dephosphorylate BAF, but their specific roles in regulating BAF phosphorylation are not yet fully understood. Here, we show that PP2A is the main phosphatase driving BAF dephosphorylation in interphase, whereas PP4 plays a minor role. During mitosis, both PP2A and PP4 localize to centromeres; however, only PP4 directly interacts with cenBAF, suggesting that it maintains this fraction in an unphosphorylated state. Notably, PP4’s centromeric localization depends on PP2A. At mitotic exit, PP2A dephosphorylates the large pool of mitotically phosphorylated BAF, while PP4 controls the timing of its re-association with chromatin. Together, these findings reveal a coordinated interplay between PP2A and PP4 in controlling BAF dephosphorylation and function.

In eukaryotes, the spatial segregation of heterochromatin and euchromatin is key for the structural organization and function of the genome. Heterochromatin interacts with the nuclear envelope (NE) and occupies a more peripheral position than euchromatin. However, the mechanisms that govern tethering of heterochromatin to the NE are not fully understood. Here, we report that Barrier-to-Autointegration Factor (BAF), a highly conserved NE-associated protein, interacts with centromeric heterochromatin and regulates its anchoring to the NE in a phosphorylation-sensitive manner. We show that impaired BAF phosphorylation leads to its persistent association with centromeric heterochromatin and reinforced anchoring. We also show that, concomitant with reinforced anchoring of centromeric heterochromatin to the NE, impaired BAF phosphorylation has important functional consequences, compromising both NE integrity and heterochromatin coalescence, and disturbing mitotic progression. Altogether, our results suggest that anchoring of centromeric heterochromatin to the NE is a highly dynamic process regulated through BAF phosphorylation, and reveal the deleterious functional consequences of perturbing this dynamic regulation.

During early embryogenesis the zygotic genome remains transcriptionally silent and expression relies on maternally deposited products. Maternal deposition of histones is crucial to preserve chromatin integrity during early embryo development, when the number of nuclei exponentially increases in the absence of zygotic expression. In the Drosophila embryo, histones are maternally deposited as both proteins and mRNAs. Histone transcripts are the only nonpolyadenylated cellular mRNAs. They contain a highly conserved 3′UTR stem-loop structure, which is recognized by the Stem-Loop Binding Protein (SLBP) that, in conjunction with U7 snRNP, regulates their unique 3′-end processing. Here we report that, unexpectedly, maternal histone mRNAs are polyadenylated and have a truncated 3′ stem-loop. This noncanonical 3′-end processing of maternal histone mRNAs occurs at their synthesis during oogenesis and requires SLBP, but not U7 snRNP. We show that maternal histone transcripts are subjected to cytoplasmic poly(A) tail elongation by Wisp, which results in their stabilization and is a requisite for translation. We also show that maternal histone transcripts remain largely quiescent and that their translation is activated upon loss of the embryonic linker histone dBigH1, which impairs chromatin assembly and induces DNA damage. Here, we discuss possible models to integrate these observations.

Chromosome pairing constitutes an important level of genome organization, yet the mechanisms that regulate pairing in somatic cells and the impact on 3D chromatin organization are still poorly understood. Here, we address these questions in Drosophila, an organism with robust somatic pairing. In Drosophila, pairing preferentially occurs at loci consisting of numerous architectural protein binding sites (APBSs), suggesting a role of architectural proteins (APs) in pairing regulation. Amongst these, the anti-pairing function of the condensin II subunit CAP-H2 is well established. However, the factors that regulate CAP-H2 localization and action at APBSs remain largely unknown. Here, we identify two factors that control CAP-H2 occupancy at APBSs and, therefore, regulate pairing. We show that Z4, interacts with CAP-H2 and is required for its localization at APBSs. We also show that hyperosmotic cellular stress induces fast and reversible unpairing in a Z4/CAP-H2 dependent manner. Moreover, by combining the opposite effects of Z4 depletion and osmostress, we show that pairing correlates with the strength of intrachromosomal 3D interactions, such as active (A) compartment interactions, intragenic gene-loops, and polycomb (Pc)-mediated chromatin loops. Altogether, our results reveal new players in CAP-H2-mediated pairing regulation and the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions.

In the nucleus, chromatin is intricately structured into multiple layers of 3D organization important for genome activity. How distinct layers influence each other is not well understood. In particular, the contribution of chromosome pairing to 3D chromatin organization has been largely neglected. Here, we address this question in Drosophila, an organism that shows robust chromosome pairing in interphasic somatic cells. The extent of chromosome pairing depends on the balance between pairing and anti-pairing factors, with the anti-pairing activity of the CAP-H2 condensin II subunit being the best documented. Here, we identify the zinc-finger protein Z4 as a strong anti-pairer that interacts with and mediates the chromatin binding of CAP-H2. We also report that hyperosmotic cellular stress induces fast and reversible chromosome unpairing that depends on Z4/CAP-H2. And, most important, by combining Z4 depletion and osmostress, we show that chromosome pairing reinforces intrachromosomal 3D interactions. On the one hand, pairing facilitates RNAPII occupancy that correlates with enhanced intragenic gene-loop interactions. In addition, acting at a distance, pairing reinforces chromatin-loop interactions mediated by Polycomb (Pc). In contrast, chromosome pairing does not affect which genomic intervals segregate to active (A) and inactive (B) compartments, with only minimal effects on the strength of A-A compartmental interactions. Altogether, our results unveil the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions, unraveling the interwoven relationship between different layers of chromatin organization and the essential contribution of chromosome pairing.

Post-translational modifications (PTMs) of core histones are important epigenetic determinants that correlate with functional chromatin states. However, despite multiple linker histone H1s PTMs have been identified, little is known about their genomic distribution and contribution to the epigenetic regulation of chromatin. Here, we address this question in Drosophila that encodes a single somatic linker histone, dH1. We previously reported that dH1 is dimethylated at K27 (dH1K27me2). Here, we show that dH1K27me2 is a major PTM of Drosophila heterochromatin. At mitosis, dH1K27me2 accumulates at pericentromeric heterochromatin, while, in interphase, it is also detected at intercalary heterochromatin. ChIPseq experiments show that >98% of dH1K27me2 enriched regions map to heterochromatic repetitive DNA elements, including transposable elements, simple DNA repeats and satellite DNAs. Moreover, expression of a mutated dH1K27A form, which impairs dH1K27me2, alters heterochromatin organization, upregulates expression of heterochromatic transposable elements and results in the accumulation of RNA:DNA hybrids (R-loops) in heterochromatin, without affecting H3K9 methylation and HP1a binding. The pattern of dH1K27me2 is H3K9 methylation independent, as it is equally detected in flies carrying a H3K9R mutation, and is not affected by depletion of Su(var)3–9, HP1a or Su(var)4–20. Altogether these results suggest that dH1K27me2 contributes to heterochromatin organization independently of H3K9 methylation.

Linker histones H1 are essential chromatin components that exist as multiple developmentally regulated variants. In metazoans, specific H1s are expressed during germline development in a tightly regulated manner. However, the mechanisms governing their stage-dependent expression are poorly understood. Here, we address this question in Drosophila , which encodes for a single germline-specific dBigH1 linker histone. We show that during female germline lineage differentiation, dBigH1 is expressed in germ stem cells and cystoblasts, becomes silenced during transit-amplifying (TA) cystocytes divisions to resume expression after proliferation stops and differentiation starts, when it progressively accumulates in the oocyte. We find that dBigH1 silencing during TA divisions is post-transcriptional and depends on the tumour suppressor Brain tumour (Brat), an essential RNA-binding protein that regulates mRNA translation and stability. Like other oocyte-specific variants, dBigH1 is maternally expressed during early embryogenesis until it is replaced by somatic dH1 at the maternal-to-zygotic transition (MZT). Brat also mediates dBigH1 silencing at MZT. Finally, we discuss the situation in testes, where Brat is not expressed, but dBigH1 is translationally silenced too.

Barrier-to-Autointegration Factor (BAF) is a conserved nuclear envelope (NE) component that binds chromatin and helps its anchoring to the NE. Cycles of phosphorylation and dephosphorylation control BAF function. Entering mitosis, phosphorylation releases BAF from chromatin and facilitates NE-disassembly. At mitotic exit, PP2A-mediated dephosphorylation restores chromatin binding and nucleates NE-reassembly. Here, we show that in Drosophila a small fraction of BAF (cenBAF) associates with centromeres. We also find that PP4 phosphatase, which is recruited to centromeres by CENP-C, prevents phosphorylation and release of cenBAF during mitosis. cenBAF is necessary for proper centromere assembly and accurate chromosome segregation, being critical for mitosis progression. Disrupting cenBAF localization prevents PP2A inactivation in mitosis compromising global BAF phosphorylation, which in turn leads to its persistent association with chromatin, delays anaphase onset and causes NE defects. These results suggest that, together with PP4 and CENP-C, cenBAF forms a centromere-based mechanism that controls chromosome segregation and mitosis progression.

In an earlier paper (Pérez-Montero et al., 2013), we reported that the embryonic linker histone of Drosophila dBigH1 was essential for early Drosophila embryogenesis since embryos homozygous for the bigH1 ¹⁰⁰ mutation showed strong defects and did not survive beyond zygotic genome activation (ZGA) at cellularization. Recent results challenge these observations since null bigH1 mutations generated by CRISPR/Cas9 methodology turn out to be homozygous viable, as reported in Li et al . (2019) and here. In this regard, Li et al . described a novel mechanism by which lack of dBigH1 is compensated by the early expression of maternal dH1. Here, we confirm this observation and show that such compensatory mechanism is not activated in bigH1 ¹⁰⁰ embryos.