A trivalent nucleosome interaction by PHIP/BRWD2 is disrupted in neurodevelopmental disorders and cancer

Mutations in the PHIP/BRWD2 chromatin regulator cause the human neurodevelopmental disorder Chung-Jansen syndrome, while alterations in PHIP expression are linked to cancer. Precisely how PHIP functions in these contexts is not fully understood. Here we demonstrate that PHIP is a chromatin-associated CRL4 ubiquitin ligase substrate receptor and is required for CRL4 recruitment to chromatin. PHIP binds to chromatin through a trivalent reader domain consisting of a H3K4-methyl binding Tudor domain and two bromodomains (BD1 and BD2). Using semisynthetic nucleosomes with defined histone post-translational modifications, we characterize PHIPs BD1 and BD2 as respective readers of H3K14ac and H4K12ac, and identify human disease-associated mutations in each domain and the intervening linker region that likely disrupt chromatin binding. These findings provide new insight into the biological function of this enigmatic chromatin protein and set the stage for the identification of both upstream chromatin modifiers and downstream targets of PHIP in human disease.

Cross-species incompatibility between a DNA satellite and a chromatin protein poisons germline genome integrity

Satellite DNA spans megabases of eukaryotic genome sequence. These vast stretches of tandem DNA repeats undergo high rates of sequence turnover, resulting in radically different satellite DNA landscapes between closely related species. Such extreme evolutionary plasticity suggests that satellite DNA accumulates mutations with no functional consequence. Paradoxically, satellite-rich genomic regions support essential, conserved nuclear processes, including chromosome segregation, dosage compensation, and nuclear structure. A leading resolution to this paradox is that deleterious alterations to satellite DNA trigger adaptive evolution of chromatin proteins to preserve these essential functions. Here we experimentally test this model of coevolution between chromatin proteins and DNA satellites by conducting an evolution-guided manipulation of both protein and satellite. We focused on an adaptively evolving, ovary-enriched chromatin protein, called Maternal Haploid (MH) from Drosophila. MH co-localizes with an 11 Mb 359-bp satellite array present in Drosophila melanogaster but absent in its sister species, D. simulans. Using CRISPR/Cas9-mediated transgenesis, we swapped the D. simulans version of MH into D. melanogaster. We discovered that D. melanogaster females encoding only the D. simulans mh (mh[sim]) do not phenocopy the mh null mutation. Instead, MH[sim] is toxic to D. melanogaster ovaries: we observed elevated ovarian cell death, reduced ovary size, and subfertility in mh[sim] females. Using both cell biological and genetic approaches, we demonstrate that MH[sim] poisons oogenesis through a DNA damage pathway. Remarkably, deleting the D. melanogaster-specific 359 satellite array from mh[sim] females completely restores female germline genome integrity and fertility. This genetic rescue offers experimental evidence that rapid evolution resulted in a cross-species incompatibility between the 359 satellite and MH. These data suggest that coevolution between ostensibly inert repetitive DNA and essential chromatin proteins preserves germline genome integrity.

SpikChIP: a novel computational methodology to compare multiple ChIP-seq using spike-in chromatin

In order to evaluate cell- and disease-specific changes in the interacting strength of chromatin targets, ChIP-seq signal across multiple conditions must undergo robust normalization. However, this is not possible using the standard ChIP-seq scheme, which lacks a reference for the control of biological and experimental variabilities. While several studies have recently proposed different solutions to circumvent this problem, substantial analytical differences among methodologies could hamper the experimental reproducibility and quantitative accuracy. Here, we propose a computational method to accurately compare ChIP-seq experiments, with exogenous spike-in chromatin, across samples in a genome-wide manner by using a local regression strategy (spikChIP). In contrast to the previous methodologies, spikChIP reduces the influence of sequencing noise of spike-in material during ChIP-seq normalization, while minimizes the overcorrection of non-occupied genomic regions in the experimental ChIP-seq. We demonstrate the utility of spikChIP with both histone and non-histone chromatin protein, allowing us to monitor for experimental reproducibility and the accurate ChIP-seq comparison of distinct experimental schemes. spikChIP software is available on GitHub (https://github.com/eblancoga/spikChIP).

TbSAP is a novel chromatin protein repressing metacyclic variant surface glycoprotein expression sites in bloodstream form Trypanosoma brucei

The African trypanosome Trypanosoma brucei is a unicellular eukaryote, which relies on a protective variant surface glycoprotein (VSG) coat for survival in the mammalian host. A single trypanosome has >2000 VSG genes and pseudogenes of which only one is expressed from one of ∼15 telomeric bloodstream form expression sites (BESs). Infectious metacyclic trypanosomes present within the tsetse fly vector also express VSG from a separate set of telomeric metacyclic ESs (MESs). All MESs are silenced in bloodstream form T. brucei. As very little is known about how this is mediated, we performed a whole genome RNAi library screen to identify MES repressors. This allowed us to identify a novel SAP domain containing DNA binding protein which we called TbSAP. TbSAP is enriched at the nuclear periphery and binds both MESs and BESs. Knockdown of TbSAP in bloodstream form trypanosomes did not result in cells becoming more ‘metacyclic-like'. Instead, there was extensive global upregulation of transcripts including MES VSGs, VSGs within the silent VSG arrays as well as genes immediately downstream of BES promoters. TbSAP therefore appears to be a novel chromatin protein playing an important role in silencing the extensive VSG repertoire of bloodstream form T. brucei.

A Functional Network of Novel Barley MicroRNAs and Their Targets in Response to Drought

The regulation of mRNA (messenger RNA) levels by microRNA-mediated activity is especially important in plant responses to environmental stresses. In this work, we report six novel barley microRNAs, including two processed from the same precursor that are severely downregulated under drought conditions. For all analyzed microRNAs, we found target genes that were upregulated under drought conditions and that were known to be involved in a plethora of processes from disease resistance to chromatin–protein complex formation and the regulation of transcription in mitochondria. Targets for novel barley microRNAs were confirmed through degradome data analysis and RT-qPCR using primers flanking microRNA-recognition site. Our results show a broad transcriptional response of barley to water deficiency conditions through microRNA-mediated gene regulation and facilitate further research on drought tolerance in crops.