Chromatin Insulator Function and Nuclear Organization

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Temporal inhibition of chromatin looping and enhancer accessibility during neuronal remodeling During development, looping of an enhancer to a promoter is frequently observed in conjunction with temporal and tissue-specific transcriptional activation. The chromatin insulator-associated protein Alan Shepard (Shep) promotes Drosophila post-mitotic neuronal remodeling by repressing transcription of master developmental regulators, such as brain tumor (brat), specifically in maturing neurons. Since insulator proteins can promote looping, we hypothesized that Shep antagonizes brat promoter interaction with an as yet unidentified enhancer. Using chromatin conformation capture and reporter assays, we identified two novel enhancer regions that increase in looping frequency with the brat promoter specifically in pupal brains after Shep depletion. The brat promoters and enhancers function independently of Shep, ruling out direct repression of these elements. Moreover, ATAC-seq in isolated neurons demonstrates that Shep restricts chromatin accessibility of a key brat enhancer as well as other enhancers genome-wide in remodeling pupal but not larval neurons. These enhancers are enriched for chromatin targets of Shep and are located at Shep-inhibited genes, suggesting direct Shep inhibition of enhancer accessibility and gene expression during neuronal remodeling. Our results provide evidence for temporal regulation of chromatin looping and enhancer accessibility during neuronal maturation. NURF301 contributes to gypsy chromatin insulator-mediated nuclear organization Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization. Isha is a su(Hw) mRNA-binding protein required for gypsy insulator function It was previously reported that the Drosophila su(Hw) mRNA physically associates with the gypsy chromatin insulator protein complex within the nucleus and may serve a noncoding function to affect insulator activity. However, how this mRNA is recruited to the gypsy complex is not known. Here we utilized RNA-affinity pull down coupled with mass spectrometry to identify a novel RNA-binding protein, Isha (CG4266), that associates with su(Hw) mRNA in vitro and in vivo. Isha harbors a conserved RNA recognition motif (RRM) and RNA Polymerase II (Pol II) C-terminal domain (CTD)-interacting domain (CID). We found that Isha physically interacts with total and elongating Pol II and associates with chromatin at the 5 end of genes in an RNA-dependent manner. Furthermore, ChIP-seq analysis reveals Isha overlaps particularly with the core gypsy insulator component CP190 on chromatin. Depletion of Isha reduces enhancer-blocking and barrier activities of the gypsy insulator and disrupts the nuclear localization of insulator bodies. Our results reveal a novel factor Isha that promotes gypsy insulator activity that may act as a nuclear RNA-binding protein adapter for su(Hw) noncoding mRNA. DiffChIPL: A differential peak analysis method for high throughput sequencing data with biological replicates based on limma ChIP-seq detects protein-DNA interactions within chromatin, such as that of chromatin structural components and transcription machinery. ChIP-seq profiles are often noisy and variable across replicates, posing a challenge to the development of effective algorithms to accurately detect differential peaks. Methods have recently been designed for this purpose but sometimes yield conflicting results that are inconsistent with the underlying biology. Most existing algorithms perform well on limited datasets. To improve differential analysis of ChIP-seq, we present a novel differential analysis method for ChIP-seq based on limma (DiffChIPL, https://github.com/yancychy/DiffChIPL). DiffChIPL is adaptive to asymmetrical or symmetrical data and can accurately report global differences. We used simulated and real datasets for transcription factor (TF) and histone modification marks to validate and benchmark our algorithm. DiffChIPL shows superior performance in sensitivity and false positive rate (FPR) in different simulations and control datasets. DiffChIPL also performs well on real ChIP-seq, CUT&RUN, CUT&Tag, and ATAC-seq datasets. DiffChIPL is an accurate and robust method, exhibiting better performance in differential analysis for a variety of applications including TF binding, histone modifications, and chromatin accessibility. Dosage compensation in Bombyx mori is achieved by partial repression of both Z chromosomes in males Interphase chromatin is organized precisely to facilitate accurate gene expression. The structure-function relationship of chromatin is epitomized in sex chromosome dosage compensation (DC), where sex-linked gene expression is balanced between males and females via sex-specific alterations to 3D chromosome structure. Studies in ZW-bearing species suggest that DC is absent or incomplete in most lineages except butterflies and moths, where male (ZZ) Z chromosome (chZ) expression is reduced by half to equal females (ZW). However, whether one chZ is inactivated (as in mammals) or both are partially repressed (as in C. elegans) is unclear. Using Oligopaints in the silkworm, Bombyx mori, we visualize autosome and chZ organization in somatic cells from both sexes for the first time. We find that B. mori interphase chromosomes are highly compact relative to Drosophila chromosomes. Importantly, we show that in B. mori males, both chZs are similar in size and shape and are more compact than autosomes or the female chZ after DC establishment, suggesting that both male chZs are partially and equally downregulated. We also find that in the early stages of DC in females, chZ chromatin becomes more accessible and Z-linked expression increases. Concomitant with these changes, the female chZ repositions toward the nuclear center, revealing the first non-sequencing-based support for Ohnos hypothesis. These studies represent the first visualization of interphase genome organization and chZ structure in Lepidoptera. We uncover intriguing similarities between DC in B. mori and C. elegans, despite these lineages harboring evolutionarily distinct sex chromosomes (ZW/XY), suggesting a possible role for holocentricity in DC mechanisms.