Control of gene expression and recombination during lymphocyte differentiation

We investigate the molecular mechanisms involved in the control of differentiation events during lymphocyte development, including antigen receptor gene expression and recombination. One approach is to characterize cis-regulatory elements and bound nuclear or transcription factors at lymphoid gene specific loci and to infer their impact on chromatin structure using molecular biology approaches and, especially, high-throughput genomics (microarrays, ChIP-Chip, ChIP-seq, RNA-seq, MeDIP, etc…) and bio-informatics; as well as transgenic mouse and gene targeting (knockout/knockin) technologies.

In parallel, transgenic and mutant animals are being used to study precursor-product relationships along lymphoid cell developmental pathways, applying molecular and cellular biology approaches and mathematical simulation [in collaboration, notably, with the team ‘Non-Linear Dynamics’ lead by R. Lima & M. Pettini at the Center for Theoretical Physics (CPT), Luminy Campus].

We also analyze the effect of epigenetic events associated with oncogenic conversion and the process of leukemogenesis; or associated with the activation of an immune response. Finally, using knockin techniques, genomics and proteomics, we intend to define novel factors and the regulatory networks that sustain lymphocyte ontogeny and activation.

Overview

Ongoing research projects: 1, 3, 6 are supervised/co-supervised by S. SPICUGLIA; 4-6 by J-C. ANDRAU; 1, 6-7 by S. JAEGER.

1- Regulation of V(D)J recombination

V(D)J recombination, the DNA rearrangement process that assembles antigen receptor genes, is required for lymphoid cell development.
We explore the molecular mechanisms involved in the regulation of T cell receptor (TCR) gene recombination during T cell differentiation, focusing on the TCRβ gene enhancer (Eβ) that commands TCRβ gene expression through the lifespan of T lymphocytes. We expect insight into enhancer specific function(s) in adaptive immunity and TCR gene allelic exclusion.

2- Epigenetic dynamics of T-cell developmentally regulated genes

Histone post-translational modifications shape the chromatin landscape. How these epigenetic tags exactly combine in order to regulate transcriptional activity during cell developmental processes is an open question.
We use primary thymocytes to investigate gene expression and epigenetic tagging by active (H3K4me1/2/3, H3K36me3) vs. repressive (H3K9me2, H3K27me3) chromatin marks, prior to and after the TCR selection checkpoints during T-cell differentiation.

3- Epigenetic characterization of cancer cells

Chromatin plays an important role in oncogenesis. Genome-wide approaches now allow large-scale characterization of epigenetic landscapes also in cancer cells.
In collaboration with researchers and clinicians from several Cancer Research Institutes and Hospitals, we define epigenetic signatures of Acute Lymphoblastic and Myeloid Leukemias and Lymphomas, with potential implications toward disease-oriented diagnosis, prognosis and therapy.

4- Basis of gene transcription during T-cell differentiation

The Carboxy-terminal-Domain (CTD) of RNA Polymerase (Pol) II harbors repetitions of a conserved heptapeptide that can be phosphorylated on Ser5 (S5P) during transcriptional initiation; on Ser2 (S2P) during elongation; and on Ser7 [S7P; characterized recently]. Likely, S5P/2P/7P combinatorial features play important roles in transcriptional controls. Enhancers may also influence the rate of transcriptional initiation via the recruitment of General Transcription Factors (GTFs) and Pol II prior to their translocation to the promoter sites.

We perform genome-wide profiling of GTFs and Pol II phosphorylated isoforms as well as various epigenetic marks, in differentiating primary T-cells. Our ChIPseq data shed light on poised areas of Pol II and the function of CTD isoforms during gene transcription. Notably, we observed Pol II and GTFs binding to known and novel enhancers in some cases resulting in local transcription. Clustering of TBP and Pol II-S5P plus RNAseq analysis indicated that the initiating transcriptional machinery spreads over large inter- and intragenic areas. We thus defined new regulatory genomic elements, which we called TIPs (Transcriptional Initiation Platforms).

Epigenetics of Lymphoid Cell Development

5- The Regulome: Genomics & proteomics of TFs in cell development and differentiation

The basis of this international program is to tag endogenous genes for mouse TFs; and to generate tagged-proteins in ES cells and in somatic cells/tissues from the derived mice. These model systems shall facilitate comparative ChIPseq and proteomic analyses to characterize TF binding sites at the genome-scale as well as the composition of TF-anchored multi-protein complexes.

6- Development of bioinformatic tools dedicated to high-throughput genomics

To assist our high-throughput genomic experiments (eg, ChIPseq, RNAseq), we develop dedicated analysis suites (eg, CoCAS, adapted to the analysis of Agilent ChiP-chip data: CoCAS (ChIP-on-Chip Analysis Suite)

7- Long-range interacting forces in biological systems

Biochemical interactions between bio-molecules are critical for cellular functions. The aqueous milieu in which these interactions normally take place theoretically opposes an electrostatic barrier, thus predicting a significant restrain in terms of interaction/reaction efficiency.

What are the physical forces that drive interaction between biological players with high specificity (in space, order and time)? With the groups led by Pr. M. PETTINI/Center for Theoretical Physics (CPT) and D. MARGUET/ CIML, we test the hypothesis that electro-dynamic forces partly sustain the catalytic power of cellular machines and long-range coherence in biological systems.