Tirtha Chakraborty

Regulation of microRNA Expression and Abundance during Lymphopoiesis

Although the cellular concentration of miRNAs is critical to their function, how miRNA expression and abundance are regulated during ontogeny is unclear. We applied miRNA-, mRNA-, and ChIP-Seq to characterize the microRNome during lymphopoiesis within the context of the transcriptome and epigenome. We show that lymphocyte-specific miRNAs are either tightly controlled by polycomb group-mediated H3K27me3 or maintained in a semi-activated epigenetic state prior to full expression. Because of miRNA biogenesis, the cellular concentration of mature miRNAs does not typically reflect transcriptional changes. However, we uncover a subset of miRNAs for which abundance is dictated by miRNA gene expression. We confirm that concentration of 5p and 3p miRNA strands depends largely on free energy properties of miRNA duplexes. Unexpectedly, we also find that miRNA strand accumulation can be developmentally regulated. Our data provide a comprehensive map of immunitys microRNome and reveal the underlying epigenetic and transcriptional forces that shape miRNA homeostasis.

miR-150 regulates the development of NK and iNKT cells.

miR-150 promotes NK cell development and interferes with iNKT cell development due to the targeting the transcription factor c-Myb.

Immunoglobulin switch μ sequence causes RNA polymerase II accumulation and reduces dA hypermutation

Repetitive DNA sequences in the immunoglobulin switch μ region form RNA-containing secondary structures and undergo hypermutation by activation-induced deaminase (AID). To examine how DNA structure affects transcription and hypermutation, we mapped the position of RNA polymerase II molecules and mutations across a 5-kb region spanning the intronic enhancer to the constant μ gene. For RNA polymerase II, the distribution was determined by nuclear run-on and chromatin immunoprecipitation assays in B cells from uracil-DNA glycosylase (UNG)–deficient mice stimulated ex vivo. RNA polymerases were found at a high density in DNA flanking both sides of a 1-kb repetitive sequence that forms the core of the switch region. The pileup of polymerases was similar in unstimulated and stimulated cells from Ung−/− and Aid−/−Ung−/− mice but was absent in cells from mice with a deletion of the switch region. For mutations, DNA was sequenced from Ung−/− B cells stimulated in vivo. Surprisingly, mutations of A nucleotides, which are incorporated by DNA polymerase η, decreased 10-fold before the repetitive sequence, suggesting that the polymerase was less active in this region. We propose that altered DNA structure in the switch region pauses RNA polymerase II and limits access of DNA polymerase η during hypermutation.

Immune Surveillance and Therapy of Lymphomas Driven by Epstein-Barr -Virus Protein LMP1 in a Mouse Model

B cells infected by Epstein-Barr virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression, EBV can spread from these cells and cause life-threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV-infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV-infected B cells in vivo, establish a preclinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a new therapeutic approach.

A 220-nucleotide deletion of the intronic enhancer reveals an epigenetic hierarchy in immunoglobulin heavy chain locus activation.

A tissue-specific transcriptional enhancer, Eμ, has been implicated in developmentally regulated recombination and transcription of the immunoglobulin heavy chain (IgH) gene locus. We demonstrate that deleting 220 nucleotides that constitute the core Eμ results in partially active locus, characterized by reduced histone acetylation, chromatin remodeling, transcription, and recombination, whereas other hallmarks of tissue-specific locus activation, such as loss of H3K9 dimethylation or gain of H3K4 dimethylation, are less affected. These observations define Eμ-independent and Eμ-dependent phases of locus activation that reveal an unappreciated epigenetic hierarchy in tissue-specific gene expression.

Localized epigenetic changes induced by DH recombination restricts recombinase to DJH junctions

Genes encoding immunoglobulin heavy chains (Igh) are assembled by rearrangement of variable (VH), diversity (DH) and joining (JH) gene segments. Three critical constraints govern VH recombination. These include timing (VH recombination follows DH recombination), precision (VH gene segments recombine only to DJH junctions) and allele specificity (VH recombination is restricted to DJH-recombined alleles). Here we provide a model for these universal features of VH recombination. Analyses of DJH-recombined alleles showed that DJH junctions were selectively epigenetically marked, became nuclease sensitive and bound RAG recombinase proteins, which thereby permitted DH-associated recombination signal sequences to initiate the second step of Igh gene assembly. We propose that VH recombination is precise, because these changes did not extend to germline DH segments located 5′ of the DJH junction.

Immunoglobulin switch µ sequence causes RNA polymerase II accumulation and reduces dA hypermutation

1. 1. Rajagopal, 2. et al . 2009. J. Exp. Med. doi: 10.1084/jem.20082514 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20082514%26rft_id%253Dinfo%253Apmid%252F19433618%26rft.genre%253Darticle%26rft_val_fmt%