Olivia I. Koues

Regulation of Acetylation at the Major Histocompatibility Complex Class II Proximal Promoter by the 19S Proteasomal ATPase Sug1

ABSTRACT Recent studies have made evident the fact that the 19S regulatory component of the proteasome has functions that extend beyond degradation, particularly in the regulation of transcription. Although 19S ATPases facilitate chromatin remodeling and acetylation events in yeast (Saccharomyces cerevisiae), it is unclear if they play similar roles in mammalian cells. We have recently shown that the 19S ATPase Sug1 positively regulates the transcription of the critical inflammatory gene for major histocompatibility complex class II (MHC-II) by stabilizing enhanceosome assembly at the proximal promoter. We now show that Sug1 is crucial for regulating histone H3 acetylation at the MHC-II proximal promoter. Sug1 binds to acetylated histone H3 and, in the absence of Sug1, histone H3 acetylation is dramatically decreased at the proximal promoter, with a preferential loss of acetylation at H3 lysine 18. Sug1 also binds to the MHC-II histone acetyltransferase CREB-binding protein (CBP) and is critical for the recruitment of CBP to the MHC-II proximal promoter. Our current study strongly implicates the 19S ATPase Sug1 in modifying histones to initiate MHC-II transcription and provides novel insights into the role of the proteasome in the regulation of mammalian transcription.

Unifying model for molecular determinants of the preselection Vβ repertoire

Significance The assembly of immunoglobulin and T-cell receptor genes by V(D)J (variable, diversity, joining) recombination must strike a balance between maximum diversification of antigen receptors and favoring gene segments with specialized functions. We quantified the use of V gene segments in the primary T-cell receptor β repertoire, defining the relative contribution of 13 parameters in shaping their recombination efficiencies. Computational analysis of these data provides a unifying model, revealing a minimal set of five parameters that predict Vβ use. This model building approach will help predict how natural alterations of large V clusters impact immune receptor repertoires. The primary antigen receptor repertoire is sculpted by the process of V(D)J recombination, which must strike a balance between diversification and favoring gene segments with specialized functions. The precise determinants of how often gene segments are chosen to complete variable region coding exons remain elusive. We quantified Vβ use in the preselection Tcrb repertoire and report relative contributions of 13 distinct features that may shape their recombination efficiencies, including transcription, chromatin environment, spatial proximity to their DβJβ targets, and predicted quality of recombination signal sequences (RSSs). We show that, in contrast to functional Vβ gene segments, all pseudo-Vβ segments are sequestered in transcriptionally silent chromatin, which effectively suppresses wasteful recombination. Importantly, computational analyses provide a unifying model, revealing a minimum set of five parameters that are predictive of Vβ use, dominated by chromatin modifications associated with transcription, but largely independent of precise spatial proximity to DβJβ clusters. This learned model-building strategy may be useful in predicting the relative contributions of epigenetic, spatial, and RSS features in shaping preselection V repertoires at other antigen receptor loci. Ultimately, such models may also predict how designed or naturally occurring alterations of these loci perturb the preselection use of variable gene segments.

Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element

Majumder et al. explore the large-scale looping architecture of the Tcrb locus early in murine thymocyte development during the generation of TCRβ diversity. They dissect novel DNA regulatory elements controlling V to D-J recombination and identify within an insulator region a distally located CTCF-containing element functioning as a tether, which facilitates looping of distal Vβ to Dβ-Jβ regions and promotes locus contraction. A second CTCF-containing element, proximal to the Dβ-Jβ region, acts as a boundary, preventing the spread of active chromatin associated with Dβ-Jβ regions. Removal of the proximal boundary element impairs the locus contraction capabilities of the tethering element.

NKG2D–NKG2D Ligand Interaction Inhibits the Outgrowth of Naturally Arising Low-Grade B Cell Lymphoma In Vivo

It is now clear that recognition of nascent tumors by the immune system is critical for survival of the host against cancer. During cancer immunoediting, the ability of the tumor to escape immune recognition is important for tumor development. The immune system recognizes tumors via the presence of classical Ags and also by conserved innate mechanisms. One of these mechanisms is the NKG2D receptor that recognizes ligands whose expression is induced by cell transformation. In this study, we show that in NKG2D receptor–deficient mice, increasing numbers of B cells begin to express NKG2D ligands as they age. Their absence in wild-type mice suggests that these cells are normally cleared by NKG2D-expressing cells. NKG2D-deficient mice and mice constitutively expressing NKG2D ligands had increased incidence of B cell tumors, confirming that the inability to clear NKG2D ligand–expressing cells was important in tumor suppression and that NKG2D ligand expression is a marker of nascent tumors. Supporting a role for NKG2D ligand expression in controlling the progression of early-stage B cell lymphomas in humans, we found higher expression of a microRNA that inhibits human NKG2D ligand expression in tumor cells from high-grade compared with low-grade follicular lymphoma patients.

Domain-Specific and Stage-Intrinsic Changes in Tcrb Conformation during Thymocyte Development

Considerable cross-talk exists between mechanisms controlling genome architecture and gene expression. AgR loci are excellent models for these processes because they are regulated at both conformational and transcriptional levels to facilitate their assembly by V(D)J recombination. Upon commitment to the double-negative stage of T cell development, Tcrb adopts a compact conformation that promotes long-range recombination between Vβ gene segments (Trbvs) and their DβJβ targets. Formation of a functional VβDβJβ join signals for robust proliferation of double-negative thymocytes and their differentiation into double-positive (DP) cells, where Trbv recombination is squelched (allelic exclusion). DP differentiation also is accompanied by decontraction of Tcrb, which has been thought to separate the entire Trbv cluster from DβJβ segments (spatial segregation-based model for allelic exclusion). However, DP cells also repress transcription of unrearranged Trbvs, which may contribute to allelic exclusion. We performed a more detailed study of developmental changes in Tcrb topology and found that only the most distal portion of the Trbv cluster separates from DβJβ segments in DP thymocytes, leaving most Trbvs spatially available for rearrangement. Preferential dissociation of distal Trbvs is independent of robust proliferation or changes in transcription, chromatin, or architectural factors, which are coordinately regulated across the entire Trbv cluster. Segregation of distal Trbvs also occurs on alleles harboring a functional VβDβJβ join, suggesting that this process is independent of rearrangement status and is DP intrinsic. Our finding that most Trbvs remain associated with DβJβ targets in DP cells revises allelic exclusion models from their current conformation-dominant to a transcription-dominant formulation.

Short-Circuiting Gene Regulatory Networks: Origins of B Cell Lymphoma

B cell lymphomas (BCLs) are characterized by widespread deregulation of gene expression compared with their normal B cell counterparts. Recent epigenomic studies defined cis-regulatory elements (REs) whose activities are altered in BCL to drive some of these pathogenic expression changes. During transformation, multiple mechanisms are employed to alter RE activities, including perturbations in the function of chromatin modifiers, which can lead to revision of the B cell epigenome. Inherited and somatic variants also alter RE function via disruption of transcription factor (TF) binding. Aberrant expression of noncoding RNAs (ncRNAs) deregulates genes involved in B cell differentiation via direct repression and post-transcriptional targeting. These discoveries have established epigenetic etiologies for B cell transformation that are being exploited in novel therapeutic approaches.

Activation of Mouse Tcrb : Uncoupling RUNX1 Function from Its Cooperative Binding with ETS1

T lineage commitment requires the coordination of key transcription factors (TFs) in multipotent progenitors that transition them away from other lineages and cement T cell identity. Two important TFs for the multipotent progenitors to T lineage transition are RUNX1 and ETS1, which bind cooperatively to composite sites throughout the genome, especially in regulatory elements for genes involved in T lymphopoiesis. Activation of the TCR β (Tcrb) locus in committed thymocytes is a critical process for continued development of these cells, and is mediated by an enhancer, Eβ, which harbors two RUNX-ETS composite sites. An outstanding issue in understanding T cell gene expression programs is whether RUNX1 and ETS1 have independent functions in enhancer activation that can be dissected from cooperative binding. We now show that RUNX1 is sufficient to activate the endogenous mouse Eβ element and its neighboring 25 kb region by independently tethering this TF without coincidental ETS1 binding. Moreover, RUNX1 is sufficient for long-range promoter-Eβ looping, nucleosome clearance, and robust transcription throughout the Tcrb recombination center, spanning both DβJβ clusters. We also find that a RUNX1 domain, termed the negative regulatory domain for DNA binding, can compensate for the loss of ETS1 binding at adjacent sites. Thus, we have defined independent roles for RUNX1 in the activation of a T cell developmental enhancer, as well as its ability to mediate specific changes in chromatin landscapes that accompany long-range induction of recombination center promoters.

Control of antigen receptor gene assembly by cis-element mediated looping

The adaptive immune system endows mammals with a sophisticated mechanism to survive in a world filled with pathogens. This dynamic defense is generated by V(D)J recombination, which enables lymphocytes to uniquely interact with an enormous range of epitopes on foreign antigens. V(D)J recombination is a set of sequentially controlled DNA cleavage and repair events, which brings variable (V), diversity (D) and joining (J) gene segments together to form functional antigen receptor genes. However, the recombination process must be stringently regulated to prevent formation of chromosomal translocations, which can lead to tumors. The process of V(D)J recombination is regulated at the level of tissue, stage and allele specificity by genetic and epigenetic mechanisms. Our lab has characterized several genetic elements that regulate chromatin accessibility and recombination at the T cell receptor beta (Tcrb) locus. These include transcriptional promoters and enhancers, which interact with each other in conformational space to form a promoter-enhancer holocomplex. The holocomplex subsequently recruits the SWI/SNF chromatin remodeling complex, leading to locus activation. However, the factors that mediate conformation and epigenetic changes required for efficient Tcrb gene assembly are unknown. We hypothesized that active transcription may stabilize the holocomplex. In order to test this, we chemically induced promoter-proximal-pausing in proT cells. We discovered that in the absence of elongating RNA polymerase II, the promoter-enhancer holocomplex is disrupted without any robust changes in transcription factor binding, structural protein recruitment or histone acetylation across the locus. Ongoing work seeks to decipher whether the transcriptional machinery is required at all for holocomplex stability. Meanwhile, in order to dissect the spatial and epigenetic mechanisms that lead to Tcrb activation, our lab has generated mutated versions of Tcrb cis-elements in a proT cell line model using Zinc Finger Nucleases. Preliminary studies on these mutants have shown that the holocomplex formation requires the enhancer (or enhancer bound elements) and also CTCF sites flanking the locus. Ongoing studies seek to identify which CTCF sites are necessary and sufficient for holocomplex stability. Using these and additional mutants, we are testing the role played by cis-elements in maintaining the chromatin and looping of Tcrb, which are essential for recombination.