Guillaume J. Filion

Systematic protein location mapping reveals five principal chromatin types in Drosophila cells

Chromatin is important for the regulation of transcription and other functions, yet the diversity of chromatin composition and the distribution along chromosomes are still poorly characterized. By integrative analysis of genome-wide binding maps of 53 broadly selected chromatin components in Drosophila cells, we show that the genome is segmented into five principal chromatin types that are defined by unique yet overlapping combinations of proteins and form domains that can extend over > 100 kb. We identify a repressive chromatin type that covers about half of the genome and lacks classic heterochromatin markers. Furthermore, transcriptionally active euchromatin consists of two types that differ in molecular organization and H3K36 methylation and regulate distinct classes of genes. Finally, we provide evidence that the different chromatin types help to target DNA-binding factors to specific genomic regions. These results provide a global view of chromatin diversity and domain organization in a metazoan cell.

A Family of Human Zinc Finger Proteins That Bind Methylated DNA and Repress Transcription

ABSTRACT In vertebrates, densely methylated DNA is associated with inactive transcription. Actors in this process include proteins of the MBD family that can recognize methylated CpGs and repress transcription. Kaiso, a structurally unrelated protein, has also been shown to bind methylated CGCGs through its three Krüppel-like C2H2 zinc fingers. The human genome contains two uncharacterized proteins, ZBTB4 and ZBTB38, that contain Kaiso-like zinc fingers. We report that ZBTB4 and ZBTB38 bind methylated DNA in vitro and in vivo. Unlike Kaiso, they can bind single methylated CpGs. When transfected in mouse cells, the proteins colocalize with foci of heavily methylated satellite DNA and become delocalized upon loss of DNA methylation. Chromatin immunoprecipitation suggests that both of these proteins specifically bind to the methylated allele of the H19/Igf2 differentially methylated region. ZBTB4 and ZBTB38 repress the transcription of methylated templates in transfection assays. The two genes have distinct tissue-specific expression patterns, but both are highly expressed in the brain. Our results reveal the existence of a family of Kaiso-like proteins that bind methylated CpGs. Like proteins of the MBD family, they are able to repress transcription in a methyl-dependent manner, yet their tissue-specific expression pattern suggests nonoverlapping functions.

Sensing X Chromosome Pairs Before X Inactivation via a Novel X-Pairing Region of the Xic

Mammalian dosage compensation involves silencing of one of the two X chromosomes in females and is controlled by the X-inactivation center (Xic). The Xic, which includes Xist and its antisense transcription unit Tsix/Xite, somehow senses the number of X chromosomes and triggers Xist up-regulation from one of the two X chromosomes in females. We found that a segment of the mouse Xic lying several hundred kilobases upstream of Xist brings the two Xics together before the onset of X inactivation. This region can autonomously drive Xic trans-interactions even as an ectopic single-copy transgene. Its introduction into male embryonic stem cells is strongly selected against, consistent with a possible role in trans-activating Xist. We propose that homologous associations driven by this novel X-pairing region (Xpr) of the Xic enable a cell to sense that more than one X chromosome is present and coordinate reciprocal Xist/Tsix expression.

The Human Enhancer Blocker CTC-binding Factor Interacts with the Transcription Factor Kaiso

CTC-binding factor (CTCF) is a DNA-binding protein of vertebrates that plays essential roles in regulating genome activity through its capacity to act as an enhancer blocker. We performed a yeast two-hybrid screen to identify protein partners of CTCF that could regulate its activity. Using full-length CTCF as bait we recovered Kaiso, a POZ-zinc finger transcription factor, as a specific binding partner. The interaction occurs through a C-terminal region of CTCF and the POZ domain of Kaiso. CTCF and Kaiso are co-expressed in many tissues, and CTCF was specifically co-immunoprecipitated by several Kaiso monoclonal antibodies from nuclear lysates. Kaiso is a bimodal transcription factor that recognizes methylated CpG dinucleotides or a conserved unmethylated sequence (TNGCAGGA, the Kaiso binding site). We identified one consensus unmethylated Kaiso binding site in close proximity to the CTCF binding site in the human 5′ β-globin insulator. We found, in an insulation assay, that the presence of this Kaiso binding site reduced the enhancer-blocking activity of CTCF. These data suggest that the Kaiso-CTCF interaction negatively regulates CTCF insulator activity.

Bayesian network analysis of targeting interactions in chromatin

In eukaryotes, many chromatin proteins together regulate gene expression. Chromatin proteins often direct the genomic binding pattern of other chromatin proteins, for example, by recruitment or competition mechanisms. The network of such targeting interactions in chromatin is complex and still poorly understood. Based on genome-wide binding maps, we constructed a Bayesian network model of the targeting interactions among a broad set of 43 chromatin components in Drosophila cells. This model predicts many novel functional relationships. For example, we found that the homologous proteins HP1 and HP1C each target the heterochromatin protein HP3 to distinct sets of genes in a competitive manner. We also discovered a central role for the remodeling factor Brahma in the targeting of several DNA-binding factors, including GAGA factor, JRA, and SU(VAR)3-7. Our network model provides a global view of the targeting interplay among dozens of chromatin components.

The inner nuclear membrane proteins Man1 and Ima1 link to two different types of chromatin at the nuclear periphery in S. pombe

Metazoan chromatin at the nuclear periphery is generally characterized by lowly expressed genes and repressive chromatin marks and presents a sub-compartment with properties distinct from the nuclear interior. To test whether the S. pombe nuclear periphery behaves similarly, we used DNA adenine methyltransferase identification (DamID) to map the target loci of two inner nuclear membrane proteins, Ima1 and Man1. We found that peripheral chromatin shows low levels of RNA-Polymerase II and nucleosome occupancy, both characteristic of repressed chromatin regions. Consistently, lowly expressed genes preferentially associate with the periphery and highly expressed genes are depleted from it. When looking at peripheral intergenic regions (IGRs), we found that divergent IGRs are enriched compared with convergent IGRs, indicating that transcription preferentially points away from the periphery rather than toward it. Interestingly, we found that Ima1 and Man1 have common, but also separate target regions in the genome. Ima1-interacting loci were enriched for the RNAi components Dcr1 and Rdp1. This agrees with previous findings that Dcr1 is localized at the nuclear periphery. In contrast, Man1 target loci were bound by the heterochromatin protein Swi6, especially at subtelomeric regions. Subtelomeric chromatin was shown to form a unique chromatin type lacking both repressive and active chromatin features and containing low levels of the histone variant H2A.Z. Thus, we find that the fission yeast nuclear periphery shows similar properties to those of metazoan cells, despite the absence of a nuclear lamina. Our results point to a role of nuclear membrane proteins in organizing chromatin domains and loops.

A Network Model of the Molecular Organization of Chromatin in Drosophila

Chromatin governs gene regulation and genome maintenance, yet a substantial fraction of the chromatin proteome is still unexplored. Moreover, a global model of the chromatin protein network is lacking. By screening >100 candidates we identify 42 Drosophila proteins that were not previously associated with chromatin, which all display specific genomic binding patterns. Bayesian network modeling of the binding profiles of these and 70 known chromatin components yields a detailed blueprint of the in vivo chromatin protein network. We demonstrate functional compartmentalization of this network, and predict functions for most of the previously unknown chromatin proteins, including roles in DNA replication and repair, and gene activation and repression.

Position effects influence HIV latency reversal

The main obstacle to curing HIV is the presence of latent proviruses in the bodies of infected patients. The partial success of reactivation therapies suggests that the genomic context of integrated proviruses can interfere with treatment. Here we developed a method called Barcoded HIV ensembles (B-HIVE) to map the chromosomal locations of thousands of individual proviruses while tracking their transcriptional activities in an infected cell population. B-HIVE revealed that, in Jurkat cells, the expression of HIV is strongest close to endogenous enhancers. The insertion site also affects the response to latency-reversing agents, because we found that phytohemagglutinin and vorinostat reactivated proviruses inserted at distinct genomic locations. From these results, we propose that combinations of drugs targeting all areas of the genome will be most effective. Overall, our data suggest that the insertion context of HIV is a critical determinant of the viral response to reactivation therapies.

Starcode: sequence clustering based on all-pairs search

Motivation: The increasing throughput of sequencing technologies offers new applications and challenges for computational biology. In many of those applications, sequencing errors need to be corrected. This is particularly important when sequencing reads from an unknown reference such as random DNA barcodes. In this case, error correction can be done by performing a pairwise comparison of all the barcodes, which is a computationally complex problem. Results: Here, we address this challenge and describe an exact algorithm to determine which pairs of sequences lie within a given Levenshtein distance. For error correction or redundancy reduction purposes, matched pairs are then merged into clusters of similar sequences. The efficiency of starcode is attributable to the poucet search, a novel implementation of the Needleman–Wunsch algorithm performed on the nodes of a trie. On the task of matching random barcodes, starcode outperforms sequence clustering algorithms in both speed and precision. Availability and implementation: The C source code is available at http://github.com/gui11aume/starcode. Contact: guillaume.filion@gmail.com

The human protein kinase HIPK2 phosphorylates and downregulates the methyl-binding transcription factor ZBTB4

HIPK2 is a eukaryotic Serine–Threonine kinase that controls cellular proliferation and survival in response to exogenous signals. Here, we show that the human transcription factor ZBTB4 is a new target of HIPK2. The two proteins interact in vitro, colocalize and associate in vivo, and HIPK2 phosphorylates several conserved residues of ZBTB4. Overexpressing HIPK2 causes the degradation of ZBTB4, whereas overexpressing a kinase-deficient mutant of HIPK2 has no effect. The chemical activation of HIPK2 also decreases the amount of ZBTB4 in cells. Conversely, the inhibition of HIPK2 by drugs or by RNA interference causes a large increase in ZBTB4 levels. This negative regulation of ZBTB4 by HIPK2 occurs under normal conditions of cell growth. In addition, the degradation is increased by DNA damage. These findings have two consequences. First, we have recently shown that ZBTB4 inhibits the transcription of p21. Therefore, the activation of p21 by HIPK2 is two-pronged: stimulation of the activator p53, and simultaneous repression of the inhibitor ZBTB4. Second, ZBTB4 is also known to bind methylated DNA and repress methylated sequences. Consequently, our findings raise the possibility that HIPK2 might influence the epigenetic regulation of gene expression at loci that remain to be identified.