| Autor: |
Ba Z; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA. zhaoqing.ba@childrens.harvard.edu.; Department of Genetics, Harvard Medical School, Boston, MA, USA. zhaoqing.ba@childrens.harvard.edu., Lou J; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.; Department of Genetics, Harvard Medical School, Boston, MA, USA., Ye AY; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.; Department of Genetics, Harvard Medical School, Boston, MA, USA., Dai HQ; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.; Department of Genetics, Harvard Medical School, Boston, MA, USA., Dring EW; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.; Department of Genetics, Harvard Medical School, Boston, MA, USA., Lin SG; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.; Department of Genetics, Harvard Medical School, Boston, MA, USA., Jain S; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.; Department of Genetics, Harvard Medical School, Boston, MA, USA., Kyritsis N; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.; Department of Genetics, Harvard Medical School, Boston, MA, USA., Kieffer-Kwon KR; Lymphocyte Nuclear Biology, NIAMS, NIH, Bethesda, MD, USA.; Center of Cancer Research, NCI, NIH, Bethesda, MD, USA., Casellas R; Lymphocyte Nuclear Biology, NIAMS, NIH, Bethesda, MD, USA. rafael.casellas@nih.gov.; Center of Cancer Research, NCI, NIH, Bethesda, MD, USA. rafael.casellas@nih.gov., Alt FW; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA. alt@enders.tch.harvard.edu.; Department of Genetics, Harvard Medical School, Boston, MA, USA. alt@enders.tch.harvard.edu. |
| Abstrakt: |
The RAG endonuclease initiates Igh locus V(D)J recombination in progenitor (pro)-B cells 1 . Upon binding a recombination centre-based J H , RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin, to locate Ds and assemble a DJ H -based recombination centre 2 . CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG scanning 3-5 ; however, their inactivation allows scanning to proximal V H s, where additional CBEs activate rearrangement and impede scanning any further upstream 5 . Distal V H utilization is thought to involve diffusional access to the recombination centre following large-scale Igh locus contraction 6-8 . Here we test the potential of linear RAG scanning to mediate distal V H usage in G1-arrested v-Abl pro-B cell lines 9 , which undergo robust D-to-J H but little V H -to-DJ H rearrangements, presumably owing to lack of locus contraction 2,5 . Through an auxin-inducible approach 10 , we degraded the cohesin component RAD21 10-12 or CTCF 12,13 in these G1-arrested lines. Degradation of RAD21 eliminated all V(D)J recombination and interactions associated with RAG scanning, except for reecombination centre-located DQ52-to-J H joining, in which synapsis occurs by diffusion 2 . Remarkably, while degradation of CTCF suppressed most CBE-based chromatin interactions, it promoted robust recombination centre interactions with, and robust V H -to-DJ H joining of, distal V H s, with patterns similar to those of 'locus-contracted' primary pro-B cells. Thus, downmodulation of CTCF-bound scanning-impediment activity promotes cohesin-driven RAG scanning across the 2.7-Mb Igh locus. |
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