Arid3b Is Critical for B Lymphocyte Development

Arid3a and Arid3b belong to a subfamily of ARID (AT-rich interaction domain) transcription factors. The Arid family is involved in regulating chromatin accessibility, proliferation, and differentiation. Arid3a and Arid3b are closely related and share a unique REKLES domain that mediates their homo- and hetero-multimerization. Arid3a was originally isolated as a B cell transcription factor binding to the AT rich matrix attachment regions (MARS) of the immunoglobulin heavy chain intronic enhancer. Deletion of Arid3a results in a highly penetrant embryonic lethality with severe defects in erythropoiesis and hematopoietic stem cells (HSCs). The few surviving Arid3a-/- (<1%) animals have decreased HSCs and early progenitors in the bone marrow, but all mature lineages are normally represented in the bone marrow and periphery except for B cells. Arid3b-/- animals die around E7.5 precluding examination of hematopoietic development. So it is unclear whether the phenotype of Arid3a loss on hematopoiesis is dependent or independent of Arid3b. In this study we circumvented this limitation by also examining hematopoiesis in mice with a conditional allele of Arid3b. Bone marrow lacking Arid3b shows decreased common lymphoid progenitors (CLPs) and downstream B cell populations while the T cell and myeloid lineages are unchanged, reminiscent of the adult hematopoietic defect in Arid3a mice. Unlike Arid3a-/- mice, HSC populations are unperturbed in Arid3b-/- mice. This study demonstrates that HSC development is independent of Arid3b, whereas B cell development requires both Arid3a and Arid3b transcription factors

Development of an online resource for recruitment research in clinical trials to organise and map current literature

Medical Research Council (MRC) Network of Hubs for Trials Methodology Research (MR/L004933/1– B2).Peer reviewedPublisher PD

The Future of the Correlated Electron Problem

The understanding of material systems with strong electron-electron interactions is the central problem in modern condensed matter physics. Despite this, the essential physics of many of these materials is still not understood and we have no overall perspective on their properties. Moreover, we have very little ability to make predictions in this class of systems. In this manuscript we share our personal views of what the major open problems are in correlated electron systems and we discuss some possible routes to make progress in this rich and fascinating field. This manuscript is the result of the vigorous discussions and deliberations that took place at Johns Hopkins University during a three-day workshop January 27, 28, and 29, 2020 that brought together six senior scientists and 46 more junior scientists. Our hope, is that the topics we have presented will provide inspiration for others working in this field and motivation for the idea that significant progress can be made on very hard problems if we focus our collective energies.Comment: 55 pages, 19 figure

The CLP Population is Significantly Decreased in Arid3b null mice.

<p>For analysis of hematopoietic populations by flow cytometry, bone marrow was harvested from <i>Arid3b</i>^<i>fl/fl</i>: <i>Mx1-Cre</i> (denoted Arid3b^-/-) mice 12 weeks after final pIpC injection. <b>A)</b> Representative FACS plots in control and <i>Arid3b</i>^<i>-/-</i> mice for analysis of CLP, ALP, and BLP populations. Control (N = 14) and <i>Arid3b</i>^<i>-/-</i> (N = 17) were examined. <b>B)</b> The CLP population was significantly decreased in <i>Arid3b</i>^<i>-/-</i> mice, and the majority of the decrease was due to decreased BLPs. <b>C)</b> Representative FACS plots in control and <i>Arid3b</i>^<i>-/-</i> mice for analysis of HSC populations. Control (N = 14) and <i>Arid3b</i>^<i>-/-</i> (N = 18) were examined. <b>D)</b> HSC populations were unchanged between control and <i>Arid3b</i>^<i>-/-</i> mice. <b>E)</b> 3 weeks after final pIpC injection, bone marrow was harvested from <i>Arid3b</i>^<i>fl/fl</i>: <i>Mx1-Cre</i> mice and lineage depleted to enrich for hematopoietic stem and progenitor cells. Cells were then cocultured with OP9 cells in the presence of IL7 and Flt3L to promote B cell development for 6 days and then analyzed by flow cytometry for B cell marker B220. Representative FACS plot of control and <i>Arid3b</i>^<i>-/-</i> cells after 6 days of coculture. <b>F)</b> OP9 experiments were run in triplicate and the decrease in B cells were statistically significant. P values determined by unpaired students t-test.</p

Arid3B regulates essential B cell developmental genes.

<p><b>A)</b> 70Z/3 Pre B cells were transduced with control (pLenti), Arid3b, (pLenti-Arid3b), Arid3a (pLenti-Arid3a), or Arid3a+Arid3b (pLenti-Arida/Arid3b) lentivirus. RNA was collected and qRT-PCR was <b>A)</b> Control, Arid3a, Arid3b, or Arid3a+Arid3b overexpressing cells were analyzed by qRT-PCR for expression of B cell genes <i>Tle4</i>, <i>Smad3</i>, <i>Nfkb1</i>, and <i>Nfkbia</i>. <b>C)</b> Primary B220+ B cells were isolated from control or <i>Arid3b</i>^<i>-/-</i> mice 3-weeks post final pIpC injection and analyzed for qRT-PCR for expression of B cell genes <i>Tle4</i>, <i>Smad3</i>, <i>Nfkb1</i>, and <i>Nfkbia</i>. P values determined by unpaired students t-test (* p < .05, ** p<0.01, *** p<0.0001).</p

Arid3b^-/- B cells produce normal serum IgG and have no defect in response to LPS during ex vivo culture.

<p><b>A)</b> Peripheral blood was collected from control (N = 4) and <i>Arid3b</i>^<i>-/-</i> (N = 5) mice via cheek bleed and analyzed for total serum IgG levels. No difference was observed between control and <i>Arid3b</i>^<i>-/-</i> mice. <b>B)</b> Splenic B cells were isolated from control (N = 3) or <i>Arid3b</i>^<i>-/-</i> (N = 3) cohorts by negative selection of CD43 (Ly-48). Cells were stained with CFSE and cultured in LPS for 72 hours before analyzing by flow cytometry. Representative plots are shown. P values determined by unpaired students t-test. <b>C)</b> No difference was observed in the percentage of proliferating cells between control and <i>Arid3b</i>^<i>-/-</i> mice. <b>D)</b> Cultures were also analyzed for their ability to differentiate into plasma cells by cell surface markers B220 and CD138. <b>E)</b> No difference was observed in the percent of short lived plasma cells between control and <i>Arid3b</i>^<i>-/-</i> cultures.</p

Loss of Arid3b in Bone Marrow Results in Decreased B cells.

<p>For analysis of hematopoietic populations by flow cytometry, bone marrow was harvested from <i>Arid3b</i>^<i>fl/fl</i>: <i>Mx1-Cre</i> (denoted Arid3b^-/-) mice 12 weeks after final pIpC injection. <b>A)</b> Representative FACS plot of B cell (B220+) and myeloid populations (CD11b+) in control or arid3b^-/- mice. Control (N = 21) and Arid3b^-/- (N = 18) were examined <b>B)</b> The decrease in B cells in <i>Arid3b</i>^<i>-/-</i> bone marrow was statistically significant. <b>C)</b> No change was observed in the myeloid populations between control and <i>Arid3b</i>^<i>-/-</i> mice. <b>D)</b> Myeloid populations were further subdivided into granulocyte (CD11b+ GR1+) and monocyte (CD11b+ GR1-) populations to assess any potential differences. Control (N = 16) and <i>Arid3b</i>^<i>-/-</i> (N = 18) were examined <b>E)</b> No difference was observed in the granulocyte population. <b>F)</b> No difference was observed in the monocyte population. Wildtype (N = 21) and <i>Arid3b</i>^<i>-/-</i> (N = 18) were examined. P values determined by unpaired students t-test.</p

Splenic B and T cell populations are unchanged in ARID3B null mice.

<p>Spleens were harvested from <i>Arid3b</i>^<i>fl/fl</i>: <i>Mx1-Cre</i> (denoted Arid3b^-/-) mice for analysis of B cell and T cell populations by flow cytometry, 12 weeks after final pIpC injection. <b>A)</b> B220+ B cells were analyzed in control (N = 11) and <i>Arid3b</i>^<i>-/-</i> (N = 10) mice. No statistically significant differences were observed in B220+ splenic B cell populations. <b>B)</b> Splenic follicular zone, marginal zone, and transitional zone B cells were evaluated using cell surface markers CD21 and IgM. Representative plots are shown. Control (N = 5) and <i>Arid3b</i>^<i>-/-</i> (N = 7) mice were analyzed. <b>C)</b> <i>Arid3b</i>^<i>-/-</i> mice had significantly decreased T1-T2 cell population with significantly increased follicular zone population. No difference was observed in marginal zone B cells. <b>D)</b> T cell populations were analyzed in spleens of control (N = 6) and Arid3b^-/- (N = 9) mice.<b>E)</b> No significant differences were observed in the CD4+ T cell populations. <b>F)</b> No significant differences were observed in CD8+ T cell populations. <b>G)</b> Control (N = 7) and <i>Arid3b</i>^<i>-/-</i> (N = 7) peritoneal cells were collected by peritoneal lavage and analyzed for cell surface expression of CD5 and B220 to identify B1 and B2 B cell subsets. <b>H)</b> A modest but significant increase was observed in the B1a population with a concomitant decrease in the B1b population, while the B2 population was unchanged. P values determined by unpaired students t-test.</p

DNA binding and transcriptional consequence of Arid3a-Arid3b interaction.

<p><b>A)</b> Co-IP of endogenous Arid3a and Arid3b in the Bcl1 murine B cell lymphoma cell line. Input, whole cell lysate; αIg, pre-immune serum; anti (α)-Arid3b mouse monoclonal antibody; detection: anti-Arid3a polyclonal antibody (details in M&M). <b>B)</b> EMSA analysis reveals Arid3a:Arid3b complex on DNA. EMSA employed anti-Arid3a and anti-Arid3b supershifts (performed at increasing concentrations; triangles) of ³²P-labeled Tx125 V_HS107 probe binding to Bcl1 nuclear proteins (details in M&M). <b>C)</b> Arid3a:Arid3b interaction results in enhanced transcription (as measured by luciferase assays) in Bcl1 mature B cells stably transfected with a MAR (Tx125 and Bf150)-containing promoter fused in frame to luciferase). Increasing amounts (0–30ug; x axis) of Arid3a and Arid3b each cloned into pcDNA3.3-TOPO were delivered by transient transfection 24 hr prior to harvest and dual luciferase activity measurement (details in M&M).</p

<i>De Novo</i> B cell production is critically impaired in Arid3b null mice.

<p>For analysis of hematopoietic populations by flow cytometry, bone marrow was harvested from <i>Arid3b</i>^<i>fl/fl</i>: <i>Mx1-Cre</i> (denoted Arid3b^-/-) mice 12 weeks after final pIpC injection. <b>A)</b> Representative FACS plots in control and Arid3b^-/- mice for analysis of pro and pre B, immature B, and recirculating B cell populations. Control (N = 16) and <i>Arid3b</i>^<i>-/-</i> (N = 9) were examined <b>B)</b> Pro and Pre B cells, as well as immature B cells, were significantly decreased in <i>Arid3b</i>^<i>-/-</i> mice. Recirculating B cells were unchanged between control and <i>Arid3b</i>^<i>-/-</i> mice. <b>C)</b> B cell populations were further analyzed using Hardy fraction cell surface markers to identify B cell populations A-F. Control (N = 6) and <i>Arid3b</i>^<i>-/-</i> (N = 7) mice were examined. <b>D)</b> Significant decreases were observed in all B cell populations of <i>Arid3b</i>^<i>-/-</i> mice, except for the C’ population. P values determined by unpaired students t-test.</p