Claudia Ribeiro de Almeida

RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination.
Ribeiro de Almeida C, Dhir S, Dhir A, Moghaddam AE, Sattentau Q, Meinhart A, Proudfoot NJ

Class switch recombination (CSR) at the immunoglobulin heavy-chain (IgH) locus is associated with the formation of R-loop structures over switch (S) regions. While these often occur co-transcriptionally between nascent RNA and template DNA, we now show that they also form as part of a post-transcriptional mechanism targeting AID to IgH S-regions. This depends on the RNA helicase DDX1 that is also required for CSR in vivo. DDX1 binds to G-quadruplex (G4) structures present in intronic switch transcripts and converts them into S-region R-loops. This in turn targets the cytidine deaminase enzyme AID to S-regions so promoting CSR. Notably R-loop levels over S-regions are diminished by chemical stabilization of G4 RNA or by the expression of a DDX1 ATPase-deficient mutant that acts as a dominant-negative protein to reduce CSR efficiency. In effect, we provide evidence for how S-region transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH locus.

Molecular cell, 70, 1097-4164, 650-662.e8, 2018

Dynamic Control of Long-Range Genomic Interactions at the Immunoglobulin κ Light-Chain Locus.
Ribeiro de Almeida C, Hendriks RW, Stadhouders R

The Igκ locus, which is spread over 3Mb of genomic DNA and contains >100 variable (V) genes, serves as an important model system to study long-range chromatin interactions. Here, we will discuss how in developing B cells in the bone marrow the accessibility of individual Vκ segments is controlled by many lineage-specific and ubiquitously expressed transcription factors that act on various cis-regulatory elements, including promoters, enhancers, and insulators. This dynamic control furthermore involves changes in subnuclear localization, histone modification, DNA demethylation, and three-dimensional locus compaction. In pro-B cells, the Igκ locus adopts a poised conformation as full contraction has been achieved and many key transcription factors already occupy the locus. Subsequently, the combined activation of pre-B cell antigen receptor signaling pathways and attenuation of IL-7R signaling in small resting pre-B cells dramatically modifies the transcription factor landscape, supporting the induction of monoallelic Igκ gene rearrangements. Hereby, the intronic and 3' Igκ enhancer elements coordinately focus their activities in the Vκ region toward frequently used Vκ genes. Recent work has drawn attention to the intriguing role of the CTCF-associated regulatory elements Cer and Sis, which are located in the Vκ-Jκ intervening region and control Igκ locus contraction and Vκ repertoire diversity. This involves CTCF-mediated locus insulation, restricting enhancer activity to the Vκ region and suppressing the preferential recombination to proximal Vκ genes. A picture emerges in which the dynamic control of long-range genomic interactions ensures correct timing of Igκ locus recombination and provides appropriate opportunities for individual Vκ gene segments to engage in Vκ-Jκ rearrangement.

Advances in immunology, 128, 1557-8445, 183-271, 2015

Pre-B cell receptor signaling induces immunoglobulin κ locus accessibility by functional redistribution of enhancer-mediated chromatin interactions.
Stadhouders R, de Bruijn MJ, Rother MB, Yuvaraj S, Ribeiro de Almeida C, Kolovos P, Van Zelm MC, van Ijcken W, Grosveld F, Soler E, Hendriks RW

During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline V(κ) transcription. To investigate whether pre-BCR signaling modulates V(κ) accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb V(κ) region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3'κ enhancer with V(κ) genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and V(κ) genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used V(κ) genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the V(κ) region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the V(κ) region, whereby the two enhancers play distinct roles.

PLoS biology, 12, 1545-7885, e1001791, 2014