Malay Mandal - Academia.edu (original) (raw)

Papers by Malay Mandal

Research paper thumbnail of A divide and conquer approach (DACA) to predict high fidelity structure of large multidomain protein BRWD1

bioRxiv (Cold Spring Harbor Laboratory), Jul 11, 2023

Therapeutic importance in inhibiting Bromodomain and WD Repeat Domain containing BRWD1 against nu... more Therapeutic importance in inhibiting Bromodomain and WD Repeat Domain containing BRWD1 against numerous human pathophysiological processes including cancers prompts prediction of a workable structure of this large protein. Here, a novel divide and conquer strategy was adopted to utilize smaller overlapping sequence-fragments of BRWD1 to further utilize their predicted structures as derived templates for prediction of complete BRWD1 structure in absence of its desired homologues in the template database. The novelty of this methodology stemmed from the requirement of templates of high sequence similarity in any comparative model based predictors whereas, the own fragments of the same target protein, BRWD1 could successfully fulfill this criteria. Additionally, the outputs of different high performing predictors including AlphaFold and RoseTTAFold were systematically integrated under the premise of Inductive Reasoning. The resultant structures were validated using existing validation parameters. Finally, a new validation paradigm was adopted to screen the best structure from the result presenting in-silico studies of known interactions of BRWD1 with various small molecules like, BD inhibitors, modified histone tails, DNA motifs and interacting proteins. The algorithm proposed in this work also paved the way for prediction of authentic structures of large size proteins.

Research paper thumbnail of BRWD1 establishes epigenetic states for germinal center initiation, maintenance, and function

Research paper thumbnail of B-1a Cells Acquire Their Unique Characteristics by Bypassing the Pre-BCR Selection Stage

Social Science Research Network, 2019

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the perit... more B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/ STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

Research paper thumbnail of B-1a Cells Acquire Their Unique Characteristics by Bypassing the Pre-BCR Selection Stage

Social Science Research Network, 2019

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the perit... more B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/ STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

Research paper thumbnail of B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage

Research paper thumbnail of BRWD1 orchestrates small pre-B cell chromatin topology by converting static to dynamic cohesin

Research paper thumbnail of STAT3 signaling in B cells controls germinal center zone organization and recycling

Cell Reports, May 1, 2023

Research paper thumbnail of Ikaros and Aiolos Inhibit Pre-B-Cell Proliferation by Directly Suppressing c-Myc Expression

Molecular and Cellular Biology, Sep 1, 2010

Pre-B-cell expansion is driven by signals from the interleukin-7 receptor and the pre-B-cell rece... more Pre-B-cell expansion is driven by signals from the interleukin-7 receptor and the pre-B-cell receptor and is dependent on cyclin D3 and c-Myc. We have shown previously that interferon regulatory factors 4 and 8 induce the expression of Ikaros and Aiolos to suppress pre-B-cell proliferation. However, the molecular mechanisms through which Ikaros and Aiolos exert their growth inhibitory effect remain to be determined. Here, we provide evidence that Aiolos and Ikaros bind to the c-Myc promoter in vivo and directly suppress c-Myc expression in pre-B cells. We further show that downregulation of c-Myc is critical for the growth-inhibitory effect of Ikaros and Aiolos. Ikaros and Aiolos also induce expression of p27 and downregulate cyclin D3 in pre-B cells, and the growth-inhibitory effect of Ikaros and Aiolos is compromised in the absence of p27. A time course analysis further reveals that downregulation of c-Myc by Ikaros and Aiolos precedes p27 induction and cyclin D3 downregulation. Moreover, downregulation of c-Myc by Ikaros and Aiolos is necessary for the induction of p27 and downregulation of cyclin D3. Collectively, our studies identify a pre-B-cell receptor signaling induced inhibitory network, orchestrated by Ikaros and Aiolos, which functions to terminate pre-B-cell expansion.

Research paper thumbnail of B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage

Nature Communications, Oct 18, 2019

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the perit... more B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/ STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

Research paper thumbnail of RAG Represents a Widespread Threat to the Lymphocyte Genome

Cell, Aug 1, 2015

Highlights d The RAG endonuclease binds to thousands of sites in the lymphocyte genome d Off-targ... more Highlights d The RAG endonuclease binds to thousands of sites in the lymphocyte genome d Off-target RAG activity is rare outside of the antigen receptor genes d Substrates for RAG cleavage are preferentially depleted at sites of RAG binding d RSSs inserted in the genome create a recombination and translocation hot spot

Research paper thumbnail of Regulation of T-cell progenitor survival and cell-cycle entry by the pre-T-cell receptor

Immunological Reviews, Feb 1, 2006

Pre-T-cell receptor (pre-TCR) functions and the study of early thymocyte development continue to ... more Pre-T-cell receptor (pre-TCR) functions and the study of early thymocyte development continue to fascinate immunologists more than 10 years after the first description and cloning of the receptor. Although multiple reports have addressed several aspects of pre-TCR signaling and function, its ability to regulate diverse functions, including proliferation, survival, and allelic exclusion of the TCR-beta locus, remains an open question. What fascinates us is its central role in the fine balance between physiological differentiation and thymocyte transformation that leads to T-cell leukemia and lymphomas. In this review, we integrate pre-TCR signaling pathways and study their effects on the regulation of T-cell progenitor cell-cycle entry and cell survival. We also connect aberrant pre-TCR signaling to deregulated proliferation and apoptotic balances and thymocyte transformation.

Research paper thumbnail of Targeting the NF-κB signaling pathway in Notch1-induced T-cell leukemia

Nature Medicine, Dec 17, 2006

Research paper thumbnail of Igβ ubiquitination activates PI3K signals required for endosomal sorting

Journal of Experimental Medicine, Nov 15, 2017

Research paper thumbnail of Regulation of lymphocyte progenitor survival by the proapoptotic activities of Bim and Bid

Proceedings of the National Academy of Sciences of the United States of America, Dec 30, 2008

On their entry into the thymus, developing lymphocyte progenitors depend on signaling from the pr... more On their entry into the thymus, developing lymphocyte progenitors depend on signaling from the pre-T cell receptor (pre-TCR), which orchestrates differentiation, cell proliferation, and survival. The exact mechanism of pre-TCR-mediated suppression of T cell death remains unclear and controversial. Here, we identify Bim and Bid, 2 members of the BH3-only group of the BCL2 family, as important regulators of pre-T cell death. Both factors are highly expressed in proapoptotic thymocytes and their expression is suppressed on signaling through the pre-TCR. Their expression is directly regulated by the transcription factors FoxO3a and p53. Bid expression and p53 activity are related to the ongoing rearrangement of the TCR loci and induced DNA damage responses. Bim expression and FoxO3a nuclear translocation are directly controlled by the pre-TCR by means of its downstream kinase Akt/PKB. Interestingly, deletion of either gene on a pre-TCR ؊/؊ background rescues survival, but fails to induce further progenitor differentiation uncoupling the 2 processes. BCL2 family ͉ cell death ͉ pre-TCR signaling ͉ T cell development Results Bim and Bid Are Overexpressed in Proapoptotic, Pre-TCR Deficient T Cell Progenitors. We have initially compared BCL2-family gene expression in pre-TCR nonexpressing, proapoptotic thymocytes, and cells that receive pre-TCR signals, suppress apoptosis, and differentiate. Thus, we have purified ''small'' (based on FSC axis), pre-TCR-nonexpressing ''DN3'' (CD25 ϩ 44-c-kit Ϫ) and compared them with ''large,'' pre-TCR-expressing ''DN4''

Research paper thumbnail of Balancing Proliferation with Igκ Recombination during B-lymphopoiesis

Frontiers in Immunology, Apr 2, 2014

The essential events of B-cell development are the stochastic and sequential rearrangement of imm... more The essential events of B-cell development are the stochastic and sequential rearrangement of immunoglobulin heavy (Igµ) and then light chain (Igκ followed by Igλ) loci. The counterpoint to recombination is proliferation, which both maintains populations of pro-B cells undergoing Igµ recombination and expands the pool of pre-B cells expressing the Igµ protein available for subsequent Igκ recombination. Proliferation and recombination must be segregated into distinct and mutually exclusive developmental stages. Failure to do so risks aberrant gene translocation and leukemic transformation. Recent studies have demonstrated that proliferation and recombination are each affected by different and antagonistic receptors.The IL-7 receptor drives proliferation while the pre-B-cell antigen receptor, which contains Igµ and surrogate light chain, enhances Igκ accessibility and recombination. Remarkably, the principal downstream proliferative effectors of the IL-7R, STAT5 and cyclin D3, directly repress Igκ accessibility through very divergent yet complementary mechanisms. Conversely, the pre-B-cell receptor represses cyclin D3 leading to cell cycle exit and enhanced Igκ accessibility.These studies reveal how cell fate decisions can be directed and reinforced at each developmental transition by single receptors. Furthermore, they identify novel mechanisms of Igκ repression that have implications for gene regulation in general.

Research paper thumbnail of Histone acetyl-lysine reader Brwd1 regulates Igκ accessibility and recombination (HEM2P.260)

Journal of Immunology, May 1, 2014

The hallmark of B lymphopoiesis is sequential rearrangement of immunoglobulin heavy (Igμ) and the... more The hallmark of B lymphopoiesis is sequential rearrangement of immunoglobulin heavy (Igμ) and the light chain (Igκ and then Igl). We recently demonstrated that downstream of IL-7R signaling, STAT5 represses Igκ transcription and recombination by recruiting Ezh2 and marking Igκ with H3K27me3 (Nat Immunol 12:1139). In addition to Igκ, there was a restricted program of genes similarly repressed by STAT5. Among these, Brwd1 which encodes a BROMO domain containing histone acetyl-lysine reader molecule, demonstrated a similar expression pattern to Igκ. Brwd1-/- mice had defective lymphopoiesis starting at the pre-B cell stage. Strikingly, there was a 10-fold reduction in Igκ recombination in Brwd1-/- small-pre B cells and this was associated with severely reduced H3K16 acetylation. Genome wide analyses of WT pre-B cells indicated that Brwd1 bound at the highest density to Igκ and other loci marked with both H3K9 acetylation and H3S10pK14 acetylation. Phosphorylation of H3S10 was dependent on pre-BCR mediated Erk activation. Interestingly, Brwd1 binding sites were highly enriched in extended GAGA motifs that, in Drosophila, are associated with Trl recruitment and enhanced chromosomal accessibility. These findings identify the epigenetic reader Brwd1 as a critical mediator of Igκ accessibility whose expression and recruitment to the Igκ locus are coordinately controlled by the IL-7R and pre-BCR.

Research paper thumbnail of BRWD1 orchestrates chromatin topology by converting static to dynamic cohesin complexes

bioRxiv (Cold Spring Harbor Laboratory), Jan 23, 2023

Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative... more Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative selection and antigen receptor gene recombination1. Transitions between each state require large, coordinated changes in epigenetic landscapes and transcriptional programs2,3. How this occurs remains unclear. Herein, we demonstrate that in small pre-B cells, the lineage and stagespecific epigenetic reader Bromodomain and WD Repeating Containing Protein 1 (BRWD1)2,4reorders three-dimensional chromatin topology to affect transition between proliferative and gene recombination molecular programs. BRWD1 regulated the switch between poised and active enhancers interacting with promoters and coordinated this withIgklocus contraction. BRWD1 did so by converting chromatin-bound static cohesin to dynamic complexes competent to mediate long-range looping. Remarkably, ATP depletion recapitulated cohesin distributions observed inBrwd1-/-cells. Therefore, in small pre-B cells, cohesin conversion is the main energetic mechanism dictating where dynamic looping occurs in the genome. Our findings provide a new mechanism of cohesin regulation and reveal how cohesin function can be dictated by lineage contextual mechanisms to facilitate specific cell fate transitions.

Research paper thumbnail of BRWD1 orchestrates chromatin topology by converting static to dynamic cohesin complexes

Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative... more Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative selection and antigen receptor gene recombination1. Transitions between each state require large, coordinated changes in epigenetic landscapes and transcriptional programs2,3. How this occurs remains unclear. Herein, we demonstrate that in small pre-B cells, the lineage and stagespecific epigenetic reader Bromodomain and WD Repeating Containing Protein 1 (BRWD1)2,4reorders three-dimensional chromatin topology to affect transition between proliferative and gene recombination molecular programs. BRWD1 regulated the switch between poised and active enhancers interacting with promoters and coordinated this withIgklocus contraction. BRWD1 did so by converting chromatin-bound static cohesin to dynamic complexes competent to mediate long-range looping. Remarkably, ATP depletion recapitulated cohesin distributions observed inBrwd1-/-cells. Therefore, in small pre-B cells, cohesin conversion is ...

Research paper thumbnail of Control of Early B Cell Development by the RNA N6-Methyladenosine Methylation

Cell Reports, 2020

The RNA N 6-methyladenosine (m 6 A) methylation is installed by the METTL3-METTL14 methyltransfer... more The RNA N 6-methyladenosine (m 6 A) methylation is installed by the METTL3-METTL14 methyltransferase complex. This modification has critical regulatory roles in various biological processes. Here, we report that deletion of Mettl14 dramatically reduces mRNA m 6 A methylation in developing B cells and severely blocks B cell development in mice. Deletion of Mettl14 impairs interleukin-7 (IL-7)-induced pro-B cell proliferation and the large-pre-B-to-small-pre-B transition and causes dramatic abnormalities in gene expression programs important for B cell development. Suppression of a group of transcripts by cytoplasmic m 6 A reader YTHDF2 is critical to the IL-7induced pro-B cell proliferation. In contrast, the block in the large-pre-B-to-small-pre-B transition is independent of YTHDF1 or YTHDF2 but is associated with a failure to properly upregulate key

Research paper thumbnail of Igβ ubiquitination activates PI3K signals required for endosomal sorting

The Journal of experimental medicine, Jan 15, 2017

A wealth of in vitro data has demonstrated a central role for receptor ubiquitination in endocyti... more A wealth of in vitro data has demonstrated a central role for receptor ubiquitination in endocytic sorting. However, how receptor ubiquitination functions in vivo is poorly understood. Herein, we report that ablation of B cell antigen receptor ubiquitination in vivo uncouples the receptor from CD19 phosphorylation and phosphatidylinositol 3-kinase (PI3K) signals. These signals are necessary and sufficient for accumulating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) on B cell receptor-containing early endosomes and proper sorting into the MHC class II antigen-presenting compartment (MIIC). Surprisingly, MIIC targeting is dispensable for T cell-dependent immunity. Rather, it is critical for activating endosomal toll-like receptors and antiviral humoral immunity. These findings demonstrate a novel mechanism of receptor endosomal signaling required for specific peripheral immune responses.

Research paper thumbnail of A divide and conquer approach (DACA) to predict high fidelity structure of large multidomain protein BRWD1

bioRxiv (Cold Spring Harbor Laboratory), Jul 11, 2023

Therapeutic importance in inhibiting Bromodomain and WD Repeat Domain containing BRWD1 against nu... more Therapeutic importance in inhibiting Bromodomain and WD Repeat Domain containing BRWD1 against numerous human pathophysiological processes including cancers prompts prediction of a workable structure of this large protein. Here, a novel divide and conquer strategy was adopted to utilize smaller overlapping sequence-fragments of BRWD1 to further utilize their predicted structures as derived templates for prediction of complete BRWD1 structure in absence of its desired homologues in the template database. The novelty of this methodology stemmed from the requirement of templates of high sequence similarity in any comparative model based predictors whereas, the own fragments of the same target protein, BRWD1 could successfully fulfill this criteria. Additionally, the outputs of different high performing predictors including AlphaFold and RoseTTAFold were systematically integrated under the premise of Inductive Reasoning. The resultant structures were validated using existing validation parameters. Finally, a new validation paradigm was adopted to screen the best structure from the result presenting in-silico studies of known interactions of BRWD1 with various small molecules like, BD inhibitors, modified histone tails, DNA motifs and interacting proteins. The algorithm proposed in this work also paved the way for prediction of authentic structures of large size proteins.

Research paper thumbnail of BRWD1 establishes epigenetic states for germinal center initiation, maintenance, and function

Research paper thumbnail of B-1a Cells Acquire Their Unique Characteristics by Bypassing the Pre-BCR Selection Stage

Social Science Research Network, 2019

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the perit... more B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/ STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

Research paper thumbnail of B-1a Cells Acquire Their Unique Characteristics by Bypassing the Pre-BCR Selection Stage

Social Science Research Network, 2019

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the perit... more B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/ STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

Research paper thumbnail of B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage

Research paper thumbnail of BRWD1 orchestrates small pre-B cell chromatin topology by converting static to dynamic cohesin

Research paper thumbnail of STAT3 signaling in B cells controls germinal center zone organization and recycling

Cell Reports, May 1, 2023

Research paper thumbnail of Ikaros and Aiolos Inhibit Pre-B-Cell Proliferation by Directly Suppressing c-Myc Expression

Molecular and Cellular Biology, Sep 1, 2010

Pre-B-cell expansion is driven by signals from the interleukin-7 receptor and the pre-B-cell rece... more Pre-B-cell expansion is driven by signals from the interleukin-7 receptor and the pre-B-cell receptor and is dependent on cyclin D3 and c-Myc. We have shown previously that interferon regulatory factors 4 and 8 induce the expression of Ikaros and Aiolos to suppress pre-B-cell proliferation. However, the molecular mechanisms through which Ikaros and Aiolos exert their growth inhibitory effect remain to be determined. Here, we provide evidence that Aiolos and Ikaros bind to the c-Myc promoter in vivo and directly suppress c-Myc expression in pre-B cells. We further show that downregulation of c-Myc is critical for the growth-inhibitory effect of Ikaros and Aiolos. Ikaros and Aiolos also induce expression of p27 and downregulate cyclin D3 in pre-B cells, and the growth-inhibitory effect of Ikaros and Aiolos is compromised in the absence of p27. A time course analysis further reveals that downregulation of c-Myc by Ikaros and Aiolos precedes p27 induction and cyclin D3 downregulation. Moreover, downregulation of c-Myc by Ikaros and Aiolos is necessary for the induction of p27 and downregulation of cyclin D3. Collectively, our studies identify a pre-B-cell receptor signaling induced inhibitory network, orchestrated by Ikaros and Aiolos, which functions to terminate pre-B-cell expansion.

Research paper thumbnail of B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage

Nature Communications, Oct 18, 2019

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the perit... more B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/ STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

Research paper thumbnail of RAG Represents a Widespread Threat to the Lymphocyte Genome

Cell, Aug 1, 2015

Highlights d The RAG endonuclease binds to thousands of sites in the lymphocyte genome d Off-targ... more Highlights d The RAG endonuclease binds to thousands of sites in the lymphocyte genome d Off-target RAG activity is rare outside of the antigen receptor genes d Substrates for RAG cleavage are preferentially depleted at sites of RAG binding d RSSs inserted in the genome create a recombination and translocation hot spot

Research paper thumbnail of Regulation of T-cell progenitor survival and cell-cycle entry by the pre-T-cell receptor

Immunological Reviews, Feb 1, 2006

Pre-T-cell receptor (pre-TCR) functions and the study of early thymocyte development continue to ... more Pre-T-cell receptor (pre-TCR) functions and the study of early thymocyte development continue to fascinate immunologists more than 10 years after the first description and cloning of the receptor. Although multiple reports have addressed several aspects of pre-TCR signaling and function, its ability to regulate diverse functions, including proliferation, survival, and allelic exclusion of the TCR-beta locus, remains an open question. What fascinates us is its central role in the fine balance between physiological differentiation and thymocyte transformation that leads to T-cell leukemia and lymphomas. In this review, we integrate pre-TCR signaling pathways and study their effects on the regulation of T-cell progenitor cell-cycle entry and cell survival. We also connect aberrant pre-TCR signaling to deregulated proliferation and apoptotic balances and thymocyte transformation.

Research paper thumbnail of Targeting the NF-κB signaling pathway in Notch1-induced T-cell leukemia

Nature Medicine, Dec 17, 2006

Research paper thumbnail of Igβ ubiquitination activates PI3K signals required for endosomal sorting

Journal of Experimental Medicine, Nov 15, 2017

Research paper thumbnail of Regulation of lymphocyte progenitor survival by the proapoptotic activities of Bim and Bid

Proceedings of the National Academy of Sciences of the United States of America, Dec 30, 2008

On their entry into the thymus, developing lymphocyte progenitors depend on signaling from the pr... more On their entry into the thymus, developing lymphocyte progenitors depend on signaling from the pre-T cell receptor (pre-TCR), which orchestrates differentiation, cell proliferation, and survival. The exact mechanism of pre-TCR-mediated suppression of T cell death remains unclear and controversial. Here, we identify Bim and Bid, 2 members of the BH3-only group of the BCL2 family, as important regulators of pre-T cell death. Both factors are highly expressed in proapoptotic thymocytes and their expression is suppressed on signaling through the pre-TCR. Their expression is directly regulated by the transcription factors FoxO3a and p53. Bid expression and p53 activity are related to the ongoing rearrangement of the TCR loci and induced DNA damage responses. Bim expression and FoxO3a nuclear translocation are directly controlled by the pre-TCR by means of its downstream kinase Akt/PKB. Interestingly, deletion of either gene on a pre-TCR ؊/؊ background rescues survival, but fails to induce further progenitor differentiation uncoupling the 2 processes. BCL2 family ͉ cell death ͉ pre-TCR signaling ͉ T cell development Results Bim and Bid Are Overexpressed in Proapoptotic, Pre-TCR Deficient T Cell Progenitors. We have initially compared BCL2-family gene expression in pre-TCR nonexpressing, proapoptotic thymocytes, and cells that receive pre-TCR signals, suppress apoptosis, and differentiate. Thus, we have purified ''small'' (based on FSC axis), pre-TCR-nonexpressing ''DN3'' (CD25 ϩ 44-c-kit Ϫ) and compared them with ''large,'' pre-TCR-expressing ''DN4''

Research paper thumbnail of Balancing Proliferation with Igκ Recombination during B-lymphopoiesis

Frontiers in Immunology, Apr 2, 2014

The essential events of B-cell development are the stochastic and sequential rearrangement of imm... more The essential events of B-cell development are the stochastic and sequential rearrangement of immunoglobulin heavy (Igµ) and then light chain (Igκ followed by Igλ) loci. The counterpoint to recombination is proliferation, which both maintains populations of pro-B cells undergoing Igµ recombination and expands the pool of pre-B cells expressing the Igµ protein available for subsequent Igκ recombination. Proliferation and recombination must be segregated into distinct and mutually exclusive developmental stages. Failure to do so risks aberrant gene translocation and leukemic transformation. Recent studies have demonstrated that proliferation and recombination are each affected by different and antagonistic receptors.The IL-7 receptor drives proliferation while the pre-B-cell antigen receptor, which contains Igµ and surrogate light chain, enhances Igκ accessibility and recombination. Remarkably, the principal downstream proliferative effectors of the IL-7R, STAT5 and cyclin D3, directly repress Igκ accessibility through very divergent yet complementary mechanisms. Conversely, the pre-B-cell receptor represses cyclin D3 leading to cell cycle exit and enhanced Igκ accessibility.These studies reveal how cell fate decisions can be directed and reinforced at each developmental transition by single receptors. Furthermore, they identify novel mechanisms of Igκ repression that have implications for gene regulation in general.

Research paper thumbnail of Histone acetyl-lysine reader Brwd1 regulates Igκ accessibility and recombination (HEM2P.260)

Journal of Immunology, May 1, 2014

The hallmark of B lymphopoiesis is sequential rearrangement of immunoglobulin heavy (Igμ) and the... more The hallmark of B lymphopoiesis is sequential rearrangement of immunoglobulin heavy (Igμ) and the light chain (Igκ and then Igl). We recently demonstrated that downstream of IL-7R signaling, STAT5 represses Igκ transcription and recombination by recruiting Ezh2 and marking Igκ with H3K27me3 (Nat Immunol 12:1139). In addition to Igκ, there was a restricted program of genes similarly repressed by STAT5. Among these, Brwd1 which encodes a BROMO domain containing histone acetyl-lysine reader molecule, demonstrated a similar expression pattern to Igκ. Brwd1-/- mice had defective lymphopoiesis starting at the pre-B cell stage. Strikingly, there was a 10-fold reduction in Igκ recombination in Brwd1-/- small-pre B cells and this was associated with severely reduced H3K16 acetylation. Genome wide analyses of WT pre-B cells indicated that Brwd1 bound at the highest density to Igκ and other loci marked with both H3K9 acetylation and H3S10pK14 acetylation. Phosphorylation of H3S10 was dependent on pre-BCR mediated Erk activation. Interestingly, Brwd1 binding sites were highly enriched in extended GAGA motifs that, in Drosophila, are associated with Trl recruitment and enhanced chromosomal accessibility. These findings identify the epigenetic reader Brwd1 as a critical mediator of Igκ accessibility whose expression and recruitment to the Igκ locus are coordinately controlled by the IL-7R and pre-BCR.

Research paper thumbnail of BRWD1 orchestrates chromatin topology by converting static to dynamic cohesin complexes

bioRxiv (Cold Spring Harbor Laboratory), Jan 23, 2023

Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative... more Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative selection and antigen receptor gene recombination1. Transitions between each state require large, coordinated changes in epigenetic landscapes and transcriptional programs2,3. How this occurs remains unclear. Herein, we demonstrate that in small pre-B cells, the lineage and stagespecific epigenetic reader Bromodomain and WD Repeating Containing Protein 1 (BRWD1)2,4reorders three-dimensional chromatin topology to affect transition between proliferative and gene recombination molecular programs. BRWD1 regulated the switch between poised and active enhancers interacting with promoters and coordinated this withIgklocus contraction. BRWD1 did so by converting chromatin-bound static cohesin to dynamic complexes competent to mediate long-range looping. Remarkably, ATP depletion recapitulated cohesin distributions observed inBrwd1-/-cells. Therefore, in small pre-B cells, cohesin conversion is the main energetic mechanism dictating where dynamic looping occurs in the genome. Our findings provide a new mechanism of cohesin regulation and reveal how cohesin function can be dictated by lineage contextual mechanisms to facilitate specific cell fate transitions.

Research paper thumbnail of BRWD1 orchestrates chromatin topology by converting static to dynamic cohesin complexes

Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative... more Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative selection and antigen receptor gene recombination1. Transitions between each state require large, coordinated changes in epigenetic landscapes and transcriptional programs2,3. How this occurs remains unclear. Herein, we demonstrate that in small pre-B cells, the lineage and stagespecific epigenetic reader Bromodomain and WD Repeating Containing Protein 1 (BRWD1)2,4reorders three-dimensional chromatin topology to affect transition between proliferative and gene recombination molecular programs. BRWD1 regulated the switch between poised and active enhancers interacting with promoters and coordinated this withIgklocus contraction. BRWD1 did so by converting chromatin-bound static cohesin to dynamic complexes competent to mediate long-range looping. Remarkably, ATP depletion recapitulated cohesin distributions observed inBrwd1-/-cells. Therefore, in small pre-B cells, cohesin conversion is ...

Research paper thumbnail of Control of Early B Cell Development by the RNA N6-Methyladenosine Methylation

Cell Reports, 2020

The RNA N 6-methyladenosine (m 6 A) methylation is installed by the METTL3-METTL14 methyltransfer... more The RNA N 6-methyladenosine (m 6 A) methylation is installed by the METTL3-METTL14 methyltransferase complex. This modification has critical regulatory roles in various biological processes. Here, we report that deletion of Mettl14 dramatically reduces mRNA m 6 A methylation in developing B cells and severely blocks B cell development in mice. Deletion of Mettl14 impairs interleukin-7 (IL-7)-induced pro-B cell proliferation and the large-pre-B-to-small-pre-B transition and causes dramatic abnormalities in gene expression programs important for B cell development. Suppression of a group of transcripts by cytoplasmic m 6 A reader YTHDF2 is critical to the IL-7induced pro-B cell proliferation. In contrast, the block in the large-pre-B-to-small-pre-B transition is independent of YTHDF1 or YTHDF2 but is associated with a failure to properly upregulate key

Research paper thumbnail of Igβ ubiquitination activates PI3K signals required for endosomal sorting

The Journal of experimental medicine, Jan 15, 2017

A wealth of in vitro data has demonstrated a central role for receptor ubiquitination in endocyti... more A wealth of in vitro data has demonstrated a central role for receptor ubiquitination in endocytic sorting. However, how receptor ubiquitination functions in vivo is poorly understood. Herein, we report that ablation of B cell antigen receptor ubiquitination in vivo uncouples the receptor from CD19 phosphorylation and phosphatidylinositol 3-kinase (PI3K) signals. These signals are necessary and sufficient for accumulating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) on B cell receptor-containing early endosomes and proper sorting into the MHC class II antigen-presenting compartment (MIIC). Surprisingly, MIIC targeting is dispensable for T cell-dependent immunity. Rather, it is critical for activating endosomal toll-like receptors and antiviral humoral immunity. These findings demonstrate a novel mechanism of receptor endosomal signaling required for specific peripheral immune responses.