Faculty Bibliography

Immunodeficiency is one of the most important causes of mortality associated with Wolf-Hirschhorn syndrome (WHS), a severe rare disease originated by a deletion in chromosome 4p. The WHS candidate 1 (WHSC1) gene has been proposed as one of the main genes responsible for many of the alterations in WHS, but its mechanism of action is still unknown. Here, we present in vivo genetic evidence showing that Whsc1 plays an important role at several points of hematopoietic development. Particularly, our results demonstrate that both differentiation and function of Whsc1-deficient B cells are impaired at several key developmental stages due to profound molecular defects affecting B cell lineage specification, commitment, fitness, and proliferation, demonstrating a causal role for WHSC1 in the immunodeficiency of WHS patients.

Immunoglobulin heavy chain (IgH) alleles have ambivalent relationships: they feature both allelic exclusion, ensuring monoallelic expression of a single immunoglobulin (Ig) allele, and frequent inter-allelic class-switch recombination (CSR) reassembling genes from both alleles. The IgH locus 3' regulatory region (3'RR) includes several transcriptional cis-enhancers promoting activation-induced cytidine deaminase (AID)-dependent somatic hypermutation (SHM) and CSR, and altogether behaves as a strong super-enhancer. It can also promote deregulated expression of translocated oncogenes during lymphomagenesis. Besides these rare, illegitimate and pathogenic interactions, we now show that under physiological conditions, the 3'RR super-enhancer supports not only legitimate cis- , but also trans-recruitment of AID, contributing to IgH inter-allelic proximity and enabling the super-enhancer on one allele to stimulate biallelic SHM and CSR. Such inter-allelic activating interactions define transvection, a phenomenon well-known in drosophila but rarely observed in mammalian cells, now appearing as a unique feature of the IgH 3'RR super-enhancer.

Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis.

B cell development is a tightly regulated process dependent on sequential rearrangements of immunoglobulin loci that encode the antigen receptor. To elucidate the role of microRNAs (miRNAs) in the orchestration of B cell development, we ablated all miRNAs at the earliest stage of B cell development by conditionally targeting the enzymes critical for RNAi in early B cell precursors. Absence of any one of these enzymes led to a block at the pro- to pre-B cell transition due to increased apoptosis and a failure of pre-B cells to proliferate. Expression of a Bcl2 transgene allowed for partial rescue of B cell development, however, the majority of the rescued B cells had low surface immunoglobulin expression with evidence of ongoing light chain editing. Our analysis revealed that miRNAs are critical for the regulation of the PTEN-AKT-FOXO1 pathway that in turn controls Rag expression during B cell development.

Use of low resolution single cell DNA FISH and population based high resolution chromosome conformation capture techniques have highlighted the importance of pairwise chromatin interactions in gene regulation. However, it is unlikely that associations involving regulatory elements act in isolation of other interacting partners that also influence their impact. Indeed, the influence of multi-loci interactions remains something of an enigma as beyond low-resolution DNA FISH we do not have the appropriate tools to analyze these. Here we present a method that uses standard 4C-seq data to identify multi-loci interactions from the same cell. We demonstrate the feasibility of our method using 4C-seq data sets that identify known pairwise and novel tri-loci interactions involving the Tcrb and Igk antigen receptor enhancers. We further show that the three Igk enhancers, MiEkappa, 3'Ekappa and Edkappa, interact simultaneously in this super-enhancer cluster, which add to our previous findings showing that loss of one element decreases interactions between all three elements as well as reducing their transcriptional output. These findings underscore the functional importance of simultaneous interactions and provide new insight into the relationship between enhancer elements. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings.

During class switch recombination (CSR), B cells replace the Igh Cmu or delta exons with another downstream constant region exon (CH), altering the antibody isotype. CSR occurs through the introduction of AID-mediated double-strand breaks (DSBs) in switch regions and subsequent ligation of broken ends. Here, we developed an assay to investigate the dynamics of DSB formation in individual cells. We demonstrate that the upstream switch region Smu is first targeted during recombination and that the mechanism underlying this control relies on 53BP1. Surprisingly, regulation of break order occurs through residual binding of 53BP1 to chromatin before the introduction of damage and independent of its established role in DNA repair. Using chromosome conformation capture, we show that 53BP1 mediates changes in chromatin architecture that affect break order. Finally, our results explain how changes in Igh architecture in the absence of 53BP1 could promote inversional rearrangements that compromise CSR.

V(D)J recombination relies on the presence of proximal enhancers that activate the antigen receptor (AgR) loci in a lineage- and stage-specific manner. Unexpectedly, we find that both active and inactive AgR enhancers cooperate to disseminate their effects in a localized and long-range manner. Here, we demonstrate the importance of short-range contacts between active enhancers that constitute an Igk super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each component. Furthermore, we establish that, in T cells, long-range contact and cooperation between the inactive Igk enhancer MiEkappa and the active Tcrb enhancer Ebeta alters enrichment of CBFbeta binding in a manner that impacts Tcrb recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer-sharing between active and inactive elements in lineage- and stage-specific control.

Imaging systems that allow visualization of specific loci and nuclear structures are highly relevant for investigating how organizational changes within the nucleus play a role in regulating gene expression and other cellular processes. Here we present a live imaging system for targeted detection of genomic regions. Our approach involves generating chimaeric transcripts of viral RNAs (MS2 and PP7) and single-guide RNAs (sgRNAs), which when co-expressed with a cleavage-deficient Cas9 can recruit fluorescently tagged viral RNA-binding proteins (MCP and PCP) to specific genomic sites. This allows for rapid, stable, low-background visualization of target loci. We demonstrate the efficiency and flexibility of our method by simultaneously labelling major and minor satellite regions as well as two individual loci on mouse chromosome 12. This system provides a tool for dual-colour labelling, which is important for tracking the dynamics of chromatin interactions and for validating epigenetic processes identified in fixed cells.

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation.