Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells

https://www.cell.com/cell/pdf/S0092-8674(15)00829-6.pdf

A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks

What determines whether a developing thymocyte becomes a CD4+ or CD8+ T cell has been an issue of longstanding debate. Here, the authors review the models that have been proposed to explain CD4/CD8-lineage choice and update us on the environmental and transcription factors that might mediate this decision.

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Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice.

Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important in B-lymphocyte development, differentiation, and signaling. Btk is a member of the Tec family of kinases. Mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Activation of Btk triggers a cascade of signaling events that culminates in the generation of calcium mobilization and fluxes, cytoskeletal rearrangements, and transcriptional regulation involving nuclear factor-kappaB (NF-kappaB) and nuclear factor of activated T cells (NFAT). In B cells, NF-kappaB was shown to bind to the Btk promoter and induce transcription, whereas the B-cell receptor-dependent NF-kappaB signaling pathway requires functional Btk. Moreover, Btk activation is tightly regulated by a plethora of other signaling proteins including protein kinase C (PKC), Sab/SH3BP5, and caveolin-1. For example, the prolyl isomerase Pin1 negatively regulates Btk by decreasing tyrosine phosphorylation and steady state levels of Btk. It is intriguing that PKC and Pin1, both of which are negative regulators, bind to the pleckstrin homology domain of Btk. To this end, we describe here novel mutations in the pleckstrin homology domain investigated for their transforming capacity. In particular, we show that the mutant D43R behaves similar to E41K, already known to possess such activity.

Bruton's tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain.

Sophisticated genetic tools have made possible the identification of the genes responsible for most well-described immunodeficiencies in the past 15 years. Mutations in Btk, components of the pre-B cell and B cell receptor (lambda5, Igalpha, Igbeta), or the scaffold protein BLNK account for approximately 90% of patients with defects in early B cell development. Hyper-IgM syndromes result from mutations in CD40 ligand, CD40, AID, or UNG in 70-80% of affected patients. Rare defects in ICOS or CD19 can result in a clinical picture that is consistent with common variable immunodeficiency, and as many as 10% of patients with this disorder have heterozygous amino acid substitutions in TACI. For all these disorders, there is considerable clinical heterogeneity in patients with the same mutation. Identifying the genetic and environmental factors that influence the clinical phenotype may enhance patient care and our understanding of normal B cell development.

Primary B Cell Immunodeficiencies: Comparisons and Contrasts

Coreceptor expression is tightly regulated during thymocyte development. Deletion of specific Cd8 enhancers leads to variegated expression of CD8αβ heterodimers in double-positive thymocytes. Here we show CD8 variegation is correlated with an epigenetic 'off' state, linking Cd8 enhancer function with chromatin remodeling of the adjacent genes Cd8a and Cd8b1 (Cd8). The zinc finger protein MAZR bound the Cd8 enhancer and interacted with the nuclear receptor corepressor N-CoR complex in double-negative thymocytes. MAZR was downregulated in double-positive and CD8 single-positive thymocytes. 'Enforced' expression of MAZR led to impaired Cd8 activation and variegated CD8 expression. Our results demonstrate epigenetic control of the Cd8 loci and identify MAZR as an important regulator of Cd8 expression.

Negative regulation of CD8 expression via CD8 enhancer-mediated recruitment of the zinc finger protein MAZR

Members of the Tec kinase family (Bmx, Btk, Itk, Rlk and Tec) are primarily expressed in the hematopoietic system and form, after the Src kinase family, the second largest class of non-receptor protein tyrosine kinases. During lymphocyte development and activation Tec kinases have important functions in signaling pathways downstream of the antigen receptors. Tec family kinases are also expressed in cells of the myeloid lineage. However, with the exception of mast cells and platelets, their biological role in the myeloid system is only poorly understood. This review summarizes the current knowledge about the function of Tec family kinases in hematopoietic cells of the myeloid lineage.

The Role of Tec Family Kinases in Myeloid Cells

Tec family kinases have important roles in lymphocytes; however, little is known about their function in monocytes/macrophages. In this study we report that Tec family kinases are essential for M-CSF (M-CSF)-induced signaling pathways that regulate macrophage survival. Compared with wild-type bone marrow-derived macrophage (BMM) cultures, Tec(-/-)Btk(-/-) BMM cultures displayed increased cell death that correlated with a severe drop in macrophage numbers. In addition, macrophages deficient in either Tec or Btk showed expression and activation of caspase-11. Elucidation of M-CSF receptor (M-CSFR) signaling pathways revealed that the total tyrosine phosphorylation pattern upon M-CSF stimulation was altered in Tec(-/-)Btk(-/-) macrophages despite normal expression and phosphorylation of the M-CSFR. Further, Tec and Btk are required for proper expression of the GM-CSF receptor alpha (GM-CSFRalpha) chain in macrophages but not dendritic cells, implicating Tec family kinases in the lineage-specific regulation of GM-CSFRalpha expression. Taken together, our study shows that Tec and Btk regulate M-CSFR signaling-induced macrophage survival and provides a novel link between Tec family kinases and the regulation of caspase-11 and GM-CSFRalpha expression.

Essential roles for the Tec family kinases Tec and Btk in M-CSF receptor signaling pathways that regulate macrophage survival.

Several developmental stage-, subset-, and lineage-specific Cd8 cis-regulatory regions have been identified. These include the E8(III) enhancer, which directs expression in double-positive (DP) thymocytes, and E8(II), which is active in DP cells and CD8(+) T cells. Using a transgenic reporter expression assay, we identified a 285-bp core fragment of the E8(III) enhancer that retains activity in DP thymocytes. In vitro characterization of the core enhancer revealed five regulatory elements that are required for full enhancer activity, suggesting that multiple factors contribute to the developmental stage-specific activity. Furthermore, deletion of E8(III) in the mouse germline showed that this enhancer is required for nonvariegated expression of CD8 in DP thymocytes when E8(II) is also deleted. These results indicate that E8(III) is one of the cis-elements that contribute to the activation of the Cd8a and Cd8b gene complex during T cell development.

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Functional and Molecular Analysis of the Double-Positive Stage-Specific CD8 Enhancer E8III during Thymocyte Development

Mast cells play crucial roles in a variety of normal and pathophysiological processes and their activation has to be tightly controlled. Here, we demonstrate that the protein tyrosine kinase Tec is a crucial regulator of murine mast cell function. Tec was activated upon Fc epsilon RI stimulation of BM-derived mast cells (BMMC). The release of histamine in the absence of Tec was normal in vitro and in vivo; however, leukotriene C(4) levels were reduced in Tec(-) (/) (-) BMMC. Furthermore, the production of IL-4 was severely impaired, and GM-CSF, TNF-alpha and IL-13 levels were also diminished. Finally, a comparison of WT, Tec(-) (/) (-), Btk(-) (/) (-) and Tec(-) (/) (-)Btk(-) (/) (-) BMMC revealed a negative role for Btk in the regulation of IL-4 production, while for the efficient production of TNF-alpha, IL-13 and GM-CSF, both Tec and Btk were required. Our results demonstrate a crucial role for Tec in mast cells, which is partially different to the function of the well-characterized family member Btk.

Bernd Unger

Papers

Negative regulation of CD8 expression via CD8 enhancer-mediated recruitment of the zinc finger protein MAZR

The Role of Tec Family Kinases in Myeloid Cells

Essential roles for the Tec family kinases Tec and Btk in M-CSF receptor signaling pathways that regulate macrophage survival.

Functional and Molecular Analysis of the Double-Positive Stage-Specific CD8 Enhancer E8III during Thymocyte Development

The protein tyrosine kinase Tec regulates mast cell function