Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation.

Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.

Biased visibility in Hi-C datasets marks dynamically regulated condensed and decondensed chromatin states genome-wide.

BACKGROUND: Proximity ligation based techniques, like Hi-C, involve restriction digestion followed by ligation of formaldehyde cross-linked chromatin. Distinct chromatin states can impact the restriction digestion, and hence the visibility in the contact maps, of engaged loci. Yet, the extent and the potential impact of digestion bias remain obscure and under-appreciated in the literature. RESULTS: Through analysis of 45 Hi-C datasets, lamina-associated domains (LADs), inactive X-chromosome in mammals, and polytene bands in fly, we first established that the DNA in condensed chromatin had lesser accessibility to restriction endonucleases used in Hi-C as compared to that in decondensed chromatin. The observed bias was independent of known systematic biases, was not appropriately corrected by existing computational methods, and needed an additional optimization step. We then repurposed this bias to identify novel condensed domains outside LADs, which were bordered by insulators and were dynamically associated with the polycomb mediated epigenetic and transcriptional states during development. CONCLUSIONS: Our observations suggest that the corrected one-dimensional read counts of existing Hi-C datasets can be reliably repurposed to study the gene-regulatory dynamics associated with chromatin condensation and decondensation, and that the existing Hi-C datasets should be interpreted with cautions.

Nonallelic transvection of multiple imprinted loci is organized by the H19 imprinting control region during germline development.

Recent observations highlight that the mammalian genome extensively communicates with itself via long-range chromatin interactions. The causal link between such chromatin cross-talk and epigenetic states is, however, poorly understood. We identify here a network of physically juxtaposed regions from the entire genome with the common denominator of being genomically imprinted. Moreover, CTCF-binding sites within the H19 imprinting control region (ICR) not only determine the physical proximity among imprinted domains, but also transvect allele-specific epigenetic states, identified by replication timing patterns, to interacting, nonallelic imprinted regions during germline development. We conclude that one locus can directly or indirectly pleiotropically influence epigenetic states of multiple regions on other chromosomes with which it interacts.

Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions.

Accumulating evidence converges on the possibility that chromosomes interact with each other to regulate transcription in trans. To systematically explore the epigenetic dimension of such interactions, we devised a strategy termed circular chromosome conformation capture (4C). This approach involves a circularization step that enables high-throughput screening of physical interactions between chromosomes without a preconceived idea of the interacting partners. Here we identify 114 unique sequences from all autosomes, several of which interact primarily with the maternally inherited H19 imprinting control region. Imprinted domains were strongly overrepresented in the library of 4C sequences, further highlighting the epigenetic nature of these interactions. Moreover, we found that the direct interaction between differentially methylated regions was linked to epigenetic regulation of transcription in trans. Finally, the patterns of interactions specific to the maternal H19 imprinting control region underwent reprogramming during in vitro maturation of embryonic stem cells. These observations shed new light on development, cancer epigenetics and the evolution of imprinting.

Convergent evolution of a genomic rearrangement may explain cancer resistance in hystrico- and sciuromorpha rodents.

The rodents of hystricomorpha and sciuromorpha suborders exhibit remarkably lower incidence of cancer. The underlying genetic basis remains obscure. We report a convergent evolutionary split of human 3p21.31, a locus hosting a large number of tumour-suppressor genes (TSGs) and frequently deleted in several tumour types, in hystrico- and sciuromorphs. Analysis of 34 vertebrate genomes revealed that the synteny of 3p21.31 cluster is functionally and evolutionarily constrained in most placental mammals, but exhibit large genomic interruptions independently in hystricomorphs and sciuromorphs, owing to relaxation of underlying constraints. Hystrico- and sciuromorphs, therefore, escape from pro-tumorigenic co-deletion of several TSGs in cis. The split 3p21.31 sub-clusters gained proximity to proto-oncogene clusters from elsewhere, which might further nullify pro-tumorigenic impact of copy number variations due to co-deletion or co-amplification of genes with opposing effects. The split of 3p21.31 locus coincided with the accelerated rate of its gene expression and the body mass evolution of ancestral hystrico- and sciuromorphs. The genes near breakpoints were associated with the traits specific to hystrico- and sciuromorphs, implying adaptive significance. We conclude that the convergently evolved chromosomal interruptions of evolutionarily constrained 3p21.31 cluster might have impacted evolution of cancer resistance, body mass variation and ecological adaptations in hystrico- and sciuromorphs.

Female-biased expression of long non-coding RNAs in domains that escape X-inactivation in mouse.

BACKGROUND: Sexual dimorphism in brain gene expression has been recognized in several animal species. However, the relevant regulatory mechanisms remain poorly understood. To investigate whether sex-biased gene expression in mammalian brain is globally regulated or locally regulated in diverse brain structures, and to study the genomic organisation of brain-expressed sex-biased genes, we performed a large scale gene expression analysis of distinct brain regions in adult male and female mice. RESULTS: This study revealed spatial specificity in sex-biased transcription in the mouse brain, and identified 173 sex-biased genes in the striatum; 19 in the neocortex; 12 in the hippocampus and 31 in the eye. Genes located on sex chromosomes were consistently over-represented in all brain regions. Analysis on a subset of genes with sex-bias in more than one tissue revealed Y-encoded male-biased transcripts and X-encoded female-biased transcripts known to escape X-inactivation. In addition, we identified novel coding and non-coding X-linked genes with female-biased expression in multiple tissues. Interestingly, the chromosomal positions of all of the female-biased non-coding genes are in close proximity to protein-coding genes that escape X-inactivation. This defines X-chromosome domains each of which contains a coding and a non-coding female-biased gene. Lack of repressive chromatin marks in non-coding transcribed loci supports the possibility that they escape X-inactivation. Moreover, RNA-DNA combined FISH experiments confirmed the biallelic expression of one such novel domain. CONCLUSION: This study demonstrated that the amount of genes with sex-biased expression varies between individual brain regions in mouse. The sex-biased genes identified are localized on many chromosomes. At the same time, sexually dimorphic gene expression that is common to several parts of the brain is mostly restricted to the sex chromosomes. Moreover, the study uncovered multiple female-biased non-coding genes that are non-randomly co-localized on the X-chromosome with protein-coding genes that escape X-inactivation. This raises the possibility that expression of long non-coding RNAs may play a role in modulating gene expression in domains that escape X-inactivation in mouse.

CTCF-Mediated Genome Architecture Regulates the Dosage of Mitotically Stable Mono-allelic Expression of Autosomal Genes.

The mechanisms that guide the clonally stable random mono-allelic expression of autosomal genes remain enigmatic. We show that (1) mono-allelically expressed (MAE) genes are assorted and insulated from bi-allelically expressed (BAE) genes through CTCF-mediated chromatin loops; (2) the cell-type-specific dynamics of mono-allelic expression coincides with the gain and loss of chromatin insulator sites; (3) dosage of MAE genes is more sensitive to the loss of chromatin insulation than that of BAE genes; and (4) inactive alleles of MAE genes are significantly more insulated than active alleles and are de-repressed upon CTCF depletion. This alludes to a topology wherein the inactive alleles of MAE genes are insulated from the spatial interference of transcriptional states from the neighboring bi-allelic domains via CTCF-mediated loops. We propose that CTCF functions as a typical insulator on inactive alleles, but facilitates transcription through enhancer-linking on active allele of MAE genes, indicating widespread allele-specific regulatory roles of CTCF.

Does genome surveillance explain the global discrepancy between binding and effect of chromatin factors?

Knocking out a chromatin factor often does not alter the transcription of its binding targets. What explains the observed disconnect between binding and effect? We hypothesize that this discrepancy could be associated with the role of chromatin factors in maintaining genetic and epigenetic integrity at promoters, and not necessarily with transcription. Through re-analysis of published datasets, we present several lines of evidence that support our hypothesis and deflate the popular assumptions. We also tested the hypothesis through mutation accumulation assays on yeast knockouts of chromatin factors. Altogether, the proposed hypothesis presents a simple explanation for the global discord between chromatin factor binding and effect. Future work in this direction might fortify the hypothesis and elucidate the underlying mechanisms.

Evolutionary Loss of Genomic Proximity to Conserved Noncoding Elements Impacted the Gene Expression Dynamics During Mammalian Brain Development.

Conserved noncoding elements (CNEs) have a significant regulatory influence on their neighboring genes. Loss of proximity to CNEs through genomic rearrangements can, therefore, impact the transcriptional states of the cognate genes. Yet, the evolutionary implications of such chromosomal alterations have not been studied. Through genome-wide analysis of CNEs and the cognate genes of representative species from five different mammalian orders, we observed a significant loss of genes' linear proximity to CNEs in the rat lineage. The CNEs and the genes losing proximity had a significant association with fetal, but not postnatal, brain development as assessed through ontology terms, developmental gene expression, chromatin marks, and genetic mutations. The loss of proximity to CNEs correlated with the independent evolutionary loss of fetus-specific upregulation of nearby genes in the rat brain. DNA breakpoints implicated in brain abnormalities of germline origin had significant representation between a CNE and the gene that exhibited loss of proximity, signifying the underlying developmental tolerance of genomic rearrangements that allowed the evolutionary splits of CNEs and the cognate genes in the rodent lineage. Our observations highlighted a nontrivial impact of chromosomal rearrangements in shaping the evolutionary dynamics of mammalian brain development and might explain the loss of brain traits, like cerebral folding of the cortex, in the rodent lineage.

Yeast PAF1 complex counters the pol III accumulation and replication stress on the tRNA genes.

The RNA polymerase (pol) III transcribes mostly short, house-keeping genes, which produce stable, non-coding RNAs. The tRNAs genes, highly transcribed by pol III in vivo are known replication fork barriers. One of the transcription factors, the PAF1C (RNA polymerase II associated factor 1 complex) is reported to associate with pol I and pol II and influence their transcription. We found low level PAF1C occupancy on the yeast pol III-transcribed genes, which is not correlated with nucleosome positions, pol III occupancy and transcription. PAF1C interacts with the pol III transcription complex and causes pol III loss from the genes under replication stress. Genotoxin exposure causes pol III but not Paf1 loss from the genes. In comparison, Paf1 deletion leads to increased occupancy of pol III, γ-H2A and DNA pol2 in gene-specific manner. Paf1 restricts the accumulation of pol III by influencing the pol III pause on the genes, which reduces the pol III barrier to the replication fork progression.

Spirulina nitrate-assimilating enzymes (NR, NiR, GS) have higher specific activities and are more stable than those of rice.

Spirulina platensis, a cyanobacterium whose N-metabolic pathway is similar to that of higher plants like rice (Oryza sativa), produces tenfold more protein, indicating a higher capacity for nitrate utilization/removal. Our in vitro analyses in crude extracts revealed that this can be attributed, at least in part, to the higher specific activities (3-6 fold) and half lives (1.2-4.4 fold) of the N-assimilating enzymes, nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) in Spirulina.

Dynamic alpha-helices: conformations that do not conform.

Structural transitions are important for the stability and function of proteins, but these phenomena are poorly understood. An extensive analysis of Protein Data Bank entries reveals 103 regions in proteins with a tendency to transform from helical to nonhelical conformation and vice versa. We find that these dynamic helices, unlike other helices, are depleted in hydrophobic residues. Furthermore, the dynamic helices have higher surface accessibility and conformational mobility (P-value = 3.35e-07) than the rigid helices. Contact analyses show that these transitions result from protein-ligand, protein-nucleic acid, and crystal-contacts. The immediate structural environment differs quantitatively (P-value = 0.003) as well as qualitatively in the two alternate conformations. Often, dynamic helix experiences more contacts in its helical conformation than in the nonhelical counterpart (P-value = 0.001). There is differential preference for the type of short contacts observed in two conformational states. We also demonstrate that the regions in protein that can undergo such large conformational transitions can be predicted with a reasonable accuracy using logistic regression model of supervised learning. Our findings have implications in understanding the molecular basis of structural transitions that are coupled with binding and are important for the function and stability of the protein. Based on our observations, we propose that several functionally relevant regions on the protein surface can switch over their conformation from coil to helix and vice-versa, to regulate the recognition and binding of their partner and hence these may work as "molecular switches" in the proteins to regulate certain biological process. Our results supports the idea that protein structure-function paradigm should transform from static to a highly dynamic one.

Structural assessment of glycyl mutations in invariantly conserved motifs.

Motifs that are evolutionarily conserved in proteins are crucial to their structure and function. In one of our earlier studies, we demonstrated that the conserved motifs occurring invariantly across several organisms could act as structural determinants of the proteins. We observed the abundance of glycyl residues in these invariantly conserved motifs. The role of glycyl residues in highly conserved motifs has not been studied extensively. Thus, it would be interesting to examine the structural perturbations induced by mutation in these conserved glycyl sites. In this work, we selected a representative set of invariant signature (IS) peptides for which both the PDB structure and mutation information was available. We thoroughly analyzed the conformational features of the glycyl sites and their local interactions with the surrounding residues. Using Ramachandran angles, we showed that the glycyl residues occurring in these IS peptides, which have undergone mutation, occurred more often in the L-disallowed as compared with the L-allowed region of the Ramachandran plot. Short range contacts around the mutation site were analyzed to study the steric effects. With the results obtained from our analysis, we hypothesize that any change of activity arising because of such mutations must be attributed to the long-range interaction(s) of the new residue if the glycyl residue in the IS peptide occurred in the L-allowed region of the Ramachandran plot. However, the mutation of those conserved glycyl residues that occurred in the L-disallowed region of the Ramachandran plot might lead to an altered activity of the protein as a result of an altered conformation of the backbone in the immediate vicinity of the glycyl residue, in addition to long range effects arising from the long side chains of the new residue. Thus, the loss of activity because of mutation in the conserved glycyl site might either relate to long range interactions or to local perturbations around the site depending upon the conformational preference of the glycyl residue.

A whole genome analysis of 5' regulatory regions of human genes for putative cis-acting modulators of nucleosome positioning.

We have carried out in silico analysis of upstream regions of 23,034 genes from the human genome for sequence motifs, which can potentially affect nucleosome positioning. Nucleosome exclusion elements (NEE) occur in 12% of the genes while less than 1% contain nucleosome positioning elements (NPE). NEE are significantly higher in 5' regions of certain categories of genes, namely, genes with active promoters, genes localised to gene-rich chromosomes 16, 17 and 19, genes having significantly higher expression levels and higher levels of occupancy of general transcription machinery proteins. NEE are also enriched in housekeeping and TATA-less genes, but are significantly under-represented in the upstream region of genes functionally classified under 'organ development' and 'morphogenesis' categories. Further, DNase I hypersensitive sites which co-localise with NEE, preferentially occur in 5' regulatory regions. Considering the positioning sequences identified so far, we speculate that low affinity nucleosome positioning in the upstream sequences of genes in the human genome is the default state requiring activation through chromatin remodelling, while, there appears to be a selection for nucleosome excluding sequences in the upstream sequences of genes that are ubiquitously expressed.

Conformational flexibility may explain multiple cellular roles of PEST motifs.

PEST sequences are one of the major motifs that serve as signal for the protein degradation and are also involved in various cellular processes such as phosphorylation and protein-protein interaction. In our earlier study, we found that these motifs contribute largely to eukaryotic protein disorder. This observation led us to evaluate their conformational variability in the nonredundant Protein Data Bank (PDB) structures. For this purpose, crystallographic temperature factors, structural alignment of multiple NMR models, and dihedral angle order parameters have been used in this study. The study has revealed the hypermobility of PEST motifs as compared to other regions of the protein. Conformational flexibility may allow them to participate in number of molecular interactions under different conditions. This analysis may explain the role of protein backbone flexibility in bringing about multiple cellular roles of PEST motifs.

Intrinsic unstructuredness and abundance of PEST motifs in eukaryotic proteomes.

The study of unfolded protein regions has gained importance because of their prevalence and important roles in various cellular functions. These regions have characteristically high net charge and low hydrophobicity. The amino acid sequence determines the intrinsic unstructuredness of a region and, therefore, efforts are ongoing to delineate the sequence motifs, which might contribute to protein disorder. We find that PEST motifs are enriched in the characterized disordered regions as compared with globular ones. Analysis of representative PDB chains revealed very few structures containing PEST sequences and the majority of them lacked regular secondary structure. A proteome-wide study in completely sequenced eukaryotes with predicted unfolded and folded proteins shows that PEST proteins make up a large fraction of unfolded dataset as compared with the folded proteins. Our data also reveal the prevalence of PEST proteins in eukaryotic proteomes (approximately 25%). Functional classification of the PEST-containing proteins shows an over- and under-representation in proteins involved in regulation and metabolism, respectively. Furthermore, our analysis shows that predicted PEST regions do not exhibit any preference to be localized in the C terminals of proteins, as reported earlier.

Spatially coordinated replication and minimization of expression noise constrain three-dimensional organization of yeast genome.

Despite recent advances, the underlying functional constraints that shape the three-dimensional organization of eukaryotic genome are not entirely clear. Through comprehensive multivariate analyses of genome-wide datasets, we show that cis and trans interactions in yeast genome have significantly distinct functional associations. In particular, (i) the trans interactions are constrained by coordinated replication and co-varying mutation rates of early replicating domains through interactions among early origins, while cis interactions are constrained by coordination of late replication through interactions among late origins; (ii)cis and trans interactions exhibit differential preference for nucleosome occupancy; (iii)cis interactions are also constrained by the essentiality and co-fitness of interacting genes. Essential gene clusters associate with high average interaction frequency, relatively short-range interactions of low variance, and exhibit less fluctuations in chromatin conformation, marking a physically restrained state of engaged loci that, we suggest, is important to mitigate the epigenetic errors by restricting the spatial mobility of loci. Indeed, the genes with lower expression noise associate with relatively short-range interactions of lower variance and exhibit relatively higher average interaction frequency, a property that is conserved across Escherichia coli,yeast, and mESCs. Altogether, our observations highlight the coordination of replication and the minimization of expression noise, not necessarily co-expression of genes, as potent evolutionary constraints shaping the spatial organization of yeast genome.

Evolutionarily conserved and conformationally constrained short peptides might serve as DNA recognition elements in intrinsically disordered regions.

Despite recent advances, it is yet not clear how intrinsically disordered regions in proteins recognize their targets without any defined structures. Short linear motifs had been proposed to mediate molecular recognition by disordered regions; however, the underlying structural prerequisite remains elusive. Moreover, the role of short linear motifs in DNA recognition has not been studied. We report a repertoire of short evolutionarily Conserved Recognition Elements (CoREs) in long intrinsically disordered regions, which have very distinct amino-acid propensities from those of known motifs, and exhibit a strong tendency to retain their three-dimensional conformations compared to adjacent regions. The majority of CoREs directly interact with the DNA in the available 3D structures, which is further supported by literature evidence, analyses of ΔΔG values of DNA-binding energies and threading-based prediction of DNA binding potential. CoREs were enriched in cancer-associated missense mutations, further strengthening their functional nature. Significant enrichment of glycines in CoREs and the preference of glycyl ϕ-Ψ values within the left-handed bridge range in the l-disallowed region of the Ramachandran plot suggest that Gly-to-nonGly mutations within CoREs might alter the backbone conformation and consequently the function, a hypothesis that we reconciled using available mutation data. We conclude that CoREs might serve as bait for DNA recognition by long disordered regions and that certain mutations in these peptides can disrupt their DNA binding potential and consequently the protein function. We further hypothesize that the preferred conformations of CoREs and of glycyl residues therein might play an important role in DNA binding. The highly ordered nature of CoREs hints at a therapeutic strategy to inhibit malicious molecular interactions using small molecules mimicking CoRE conformations.

Did the modulation of expression noise shape the evolution of three dimensional genome organizations in eukaryotes?

The pervasive role of distant chromatin interactions in transcriptional regulation is increasingly becoming evident. There is a possibility that the greater diversity in chromatin interactions of a genomic locus could contribute to stochastic variation in its gene expression. However, this issue has not been addressed. Here, I present a few lines of evidence, which suggest that the variation in trans chromatin interactions might occur at the cost of expression noise. Genomic regions with nucleosome depletion, abundant and rapid transcription and with essential gene clusters exhibit relatively fewer trans chromatin interactions in the nucleus. Moreover, loci with greater number of interactions tend to show higher expression noise. Based on these observations, I hypothesize that the three dimensional organization of eukaryotic genomes might have evolved under a selective pressure to minimize the expression noise of essential gene clusters in the nucleus.

Disordered proteins and network disorder in network descriptions of protein structure, dynamics and function: hypotheses and a comprehensive review.

During the last decade, network approaches became a powerful tool to describe protein structure and dynamics. Here we review the links between disordered proteins and the associated networks, and describe the consequences of local, mesoscopic and global network disorder on changes in protein structure and dynamics. We introduce a new classification of protein networks into 'cumulus-type', i.e., those similar to puffy (white) clouds, and 'stratus-type', i.e., those similar to flat, dense (dark) low-lying clouds, and relate these network types to protein disorder dynamics and to differences in energy transmission processes. In the first class, there is limited overlap between the modules, which implies higher rigidity of the individual units; there the conformational changes can be described by an 'energy transfer' mechanism. In the second class, the topology presents a compact structure with significant overlap between the modules; there the conformational changes can be described by 'multi-trajectories'; that is, multiple highly populated pathways. We further propose that disordered protein regions evolved to help other protein segments reach 'rarely visited' but functionally-related states. We also show the role of disorder in 'spatial games' of amino acids; highlight the effects of intrinsically disordered proteins (IDPs) on cellular networks and list some possible studies linking protein disorder and protein structure networks.