[Show abstract][Hide abstract]ABSTRACT: During the early development of the gastrointestinal tract, signaling through the receptor tyrosine kinase RET is required for initiation of lymphoid organ (Peyer's patch) formation and for intestinal innervation by enteric neurons. RET signaling occurs through glial cell line-derived neurotrophic factor (GDNF) family receptor α co-receptors present in the same cell (signaling in cis). It is unclear whether RET signaling in trans, which occurs in vitro through co-receptors from other cells, has a biological role. We showed that the initial aggregation of hematopoietic cells to form lymphoid clusters occurred in a RET-dependent, chemokine-independent manner through adhesion-mediated arrest of lymphoid tissue initiator (LTin) cells. Lymphoid tissue inducer cells were not necessary for this initiation phase. LTin cells responded to all RET ligands in trans, requiring factors from other cells, whereas RET was activated in enteric neurons exclusively by GDNF in cis. Furthermore, genetic and molecular approaches revealed that the versatile RET responses in LTin cells were determined by distinct patterns of expression of the genes encoding RET and its co-receptors. Our study shows that a trans RET response in LTin cells determines the initial phase of enteric lymphoid organ morphogenesis, and suggests that differential co-expression of Ret and Gfra can control the specificity of RET signaling.
[Show abstract][Hide abstract]ABSTRACT: Hox code of K562 leukemic cell line. ChIP-seq data of histone H3 trimethylation at K4, K27, and K36 and RNA polymerase II developed by Broad Institute were obtained from the ENCODE database. The raw data were mapped to the human genome GRCh37/hg19 assembly using bwa and peak detection was carried out using MACS. Presence or absence of activation marks (K4me3, K36me3, and Pol2) and a repression mark (K27me3) indicate that subsets of HoxB and HoxC genes are transcribed in K562 cells whereas HoxA and HoxD loci are largely silent.
[Show abstract][Hide abstract]ABSTRACT: ChIP analysis of the HoxC8 promoter using antibodies against the C-terminal end of ASH1. ChIP analysis was carried out as in Fig. 1B using house-made rabbit polyclonal antibodies against the C-terminal epitope of ASH1. Preferential bindings of ASH1 to the promoter as well as downstream region of HoxC8 are shown.
[Show abstract][Hide abstract]ABSTRACT: Effects of ASH1 and MLL1 knockdown on haematopoiesis. Effects of ASH1 and MLL1 knockdown on haematopoietic development in vivo. Purified haematopoietic stem cells were transduced with lentiviral vectors expressing control GFP (closed bars) or shRNA for ASH1 (hashed bars) or MLL1(open bars) and transplanted into sublethally irradiated mice. Donor-derived cells were distinguished from host cells by Ly-5.1/Ly-5.2 allotypes, and proportions of lineage marker-positive cells among donor-derived cells are indicated. Knockdown of MLL1 caused statistically significant (p<0.05) reduction in the number of cells expressing CD11b (macrophages) or Gr-1 (granulocytes) and reciprocal increase in the number of lymphoid cells expressing CD19. The effects of ASH1 knockdown were not statistically significant but were similar to those of MLL1 knockdown.
[Show abstract][Hide abstract]ABSTRACT: Hox genes play important roles in haematopoietic development in mammals. ASH1 is a member of the trithorax group (trxG) that is required for proper expression of Hox genes and is preferentially expressed in haematopoietic stem cells. We have recently reported that ASH1 methylates histone H3 at lysine 36 (K36) but its biological function has remained elusive. Here we show that ASH1 regulates Hox gene expression positively and negatively in a leukemic cell line K562 and is required for myelomonocytic differentiation of murine haematopoietic stem cells. ASH1 binds to endogenous Hox loci in K562 cells and its knockdown causes reduced expression of Hox genes. In addition, ASH1 and MLL1 induce more than 100-fold activation of Hox promoters in HeLa cells if expressed simultaneously but not individually. Notably, ASH1 harbouring a point mutation that kills methyltransferase activity is more efficient than wild type ASH1 in Hox gene activation, indicating that K36 methylation is not a prerequisite for Hox gene expression. Moreover, tethering wild type or catalytically inactive methyltransferase domain of ASH1 to a heterologous promoter causes downregulation or upregulation, respectively, of transcription, supporting a hypothesis that K36 methylation imparts repression. Knockdown of ASH1 in K562 cells in vitro causes increased expression of ε-globin gene and reduced expression of myelomonocytic markers GPIIb and GPIIIa, whereas knockdown of ASH1 in murine haematopoietic stem cells in vivo results in decreased number of macrophages and granulocytes, a phenotype similar to that induced by loss of mll1 function. Taken together, our data suggest that ASH1 and MLL1 synergize in activation of Hox genes and thereby regulate development of myelomonocytic lineages from haematopoietic stem cells.
[Show abstract][Hide abstract]ABSTRACT: Hox promoter is sensitive to low dose okadaic acid. HeLa cells were transfected with HoxA9-firefly luciferase (open squares) and CMV-renilla luciferase (closed squares) vectors together with ASH1 and MLL1 expression vectors (1 µg each) in the presence of absence of okadaic acid. Okadaic acid enhances transcription of HoxA9 but CMV promoter at a concentration of 1 nM which is known to inhibit protein phosphatase 2A but not protein phosphatase 1A, suggesting that Hox promoter activation by ASH1 and MLL1 is sensitive to the activity of protein phosphatase 2A.
[Show abstract][Hide abstract]ABSTRACT: Titration of ASH1 and MLL1 in Hox gene activation. HeLa cells were transfected with either fixed amount (1 µg) of MLL1 and different amount of ASH1 expression vectors (closed circles) or fixed amount (1 µg) of ASH1 and different amount of MLL1 (open circles) together with HoxA9-luciferase reporter. Luciferase activities corrected by CMV-renilla luciferase activities are plotted. ASH1 and MLL1 shows first order and seconder order reaction kinetics, respectively.
[Show abstract][Hide abstract]ABSTRACT: Deletion mutants of ASH1. Despite the clear evidence that full-length ASH1 has a strong transactivation potential, it is hardly detectable by Western blot analysis, . We constructed a series of deletion mutants of ASH1 to identify fragments which can be detected and quantified by Western blot. Mutants in group (A) are not detectable and those in group (B) are detectable by Western blot. Inclusion of region III and either region I or region II of the N-terminal part of ASH1 appears to render proteins invisible by Western blot. The largest ASH1 mutant that can be identified at a protein level is ASH1Δ216-1283 and it has a Hox promoter activation potential comparable to that of wild type ASH1 (Fig. 2C).
[Show abstract][Hide abstract]ABSTRACT: Hox promoter-luciferase reporter experiments. Raw readings of firefly and renilla luciferase activities using Dual-Luciferase Reporter Assay System (Promega).
[Show abstract][Hide abstract]ABSTRACT: The human CD2 (hCD2) locus control region (LCR) inserted in the mouse CD8 gene complex activates expression of the CD8 genes in T cell subsets in which the CD8 locus is normally silenced (e.g., CD4(+) single-positive T cells). In this article, we show that, in conditional mCD8/hCD2-LCR (CD8/LCR) knock-in mice, the continuous presence of the hCD2-LCR is required for this effect. Deletion of the inserted hCD2-LCR in a developmental stage and cell lineage-specific manner revealed that the temporary presence of the LCR during early development does not permanently alter the expression pattern of the CD8 genes. As a result, cells that have been affected by the insertion of the LCR can convert to their destined phenotype once the LCR is removed. DNaseI hypersensitive sites 1 and 2 of the hCD2-LCR influence the expression of the CD8 genes in a similar manner as does the full LCR, whereas insertion of hypersensitive site 3 alone of the LCR does not result in a changed expression pattern. This analysis revealed a dynamic interaction between the hCD2-LCR and the endogenous regulatory elements of the CD8 genes.
[Show abstract][Hide abstract]ABSTRACT: During cortical development, coordination of proliferation and differentiation ensures the timely generation of different neural progenitor lineages that will give rise to mature neurons and glia. Geminin is an inhibitor of DNA replication and it has been proposed to regulate cell proliferation and fate determination during neurogenesis via interactions with transcription factors and chromatin remodeling complexes. To investigate the in vivo role of Geminin in the maintenance and differentiation of cortical neural progenitors, we have generated mice that lack Geminin expression in the developing cortex. Our results show that loss of Geminin leads to the expansion of neural progenitor cells located at the ventricular and subventricular zones of the developing cortex. Early cortical progenitors lacking Geminin exhibit a longer S-phase and a reduced ability to generate early born neurons, consistent with a preference on self-renewing divisions. Overexpression of Geminin in progenitor cells of the cortex reduces the number of neural progenitor cells, promotes cell cycle exit and subsequent neuronal differentiation. Our study suggests that Geminin has an important role during cortical development in regulating progenitor number and ultimately neuron generation.
[Show abstract][Hide abstract]ABSTRACT: The CD8 gene is silent in CD4(-)CD8(-) double-negative thymocytes, expressed in CD4(+)CD8(+) double-positive cells, and silenced in cells committing to the CD4(+) single-positive (SP) lineage, remaining active in the CD8(+) SP lineage. In this study, we show that the chromatin of the CD8 locus is remodeled in C57BL/6 and B6/J Rag1(-/-) MOM double-negative thymocytes as indicated by DNaseI hypersensitivity and widespread bivalent chromatin marks. Pre-TCR signaling coincides with chromatin bivalency resolution into monovalent activating modifications in double-positive and CD8 SP cells. Shortly after commitment to CD4 SP cell lineage, monovalent repressive characteristics and chromatin inaccessibility are established. Differential binding of Ikaros, NuRD, and heterochromatin protein 1α on the locus during these processes may participate in the complex regulation of CD8.
Full-text Article · Jun 2011 · The Journal of Immunology
[Show abstract][Hide abstract]ABSTRACT: Thymic T cell differentiation to peripheral T cells aims to assist the generation of effector cells mediating adaptive immune responses. During this process, which takes place during embryogenesis and in adulthood, proliferation is coupled with changes in chromatin organization and transcription. Moreover, B and T lymphocytes start to proliferate and rapidly expand their numbers when activated following an encounter with an antigen. This expansion phase is accompanied by differentiation of naïve T cells and is followed by a period of population contraction, resulting in only a small fraction of the expanded population surviving and entering the memory cell pool. The kinetics of the expansion and contraction affect the speed of antigen clearance and the clinical course of disease. Molecules that are involved in the coordination of proliferation, chromatin reorganization, and transcriptional regulation are likely to play an important role in T cell generation, homeostasis, and disease. Here we review how cell cycle regulators affect lymphoid system development and homeostasis and discuss recent evidence implicating the cell cycle inhibitor Geminin in this process. Geminin has been shown to coordinate proliferation and differentiation by regulating cell cycle progression, chromatin organization, and transcription in the nervous system. In the immune system, progenitor T cell commitment and differentiation progresses normally in the absence of Geminin. However, Geminin is required for TCR response in vitro and T cell proliferation upon lymphopenia-induced proliferation, suggesting that Geminin might be an essential factor for T cell expansion during the immune response.
Full-text Article · Jan 2011 · Critical Reviews in Immunology
[Show abstract][Hide abstract]ABSTRACT: This chapter describes the evolution of lymph nodes (LNs) allowing for the establishment of efficient immune responses. LNs are among the key evolutionary advantages that have made mammals so successful, allowing the establishment of protective memory T cell responses and long-term immunity in times of crisis. The correlation shown in this chapter between the evolution of lymphotoxin, LNs, and T cell memory provides a compelling potential connection of how structure and function co-evolved in the immune system. Lymphoid tissue inducer (LTi) cells are crucial for secondary lymphoid organ development in a lymphotoxin dependent manner. A series of recent published papers have shown that in adulthood LTi cells mediate key functions in innate responses to enteric pathogens, suggesting an important role for these cells in the maintenance of epithelial barriers. In the adult, LTi cells accumulate in enteric cryptal patches that develop into isolated lymphoid follicles (ILF) in response to the commensal flora. Thus, in evolutionary terms, LTi innate immune functions may have preceded their LTi potential.
Article · Dec 2010 · Progress in molecular biology and translational science
[Show abstract][Hide abstract]ABSTRACT: To study the influence of a locus control region (LCR) on the expression of a highly characterized, developmentally regulated locus, we have targeted the hCD2-LCR as a single copy into the endogenous mouse CD8 gene complex. Two knock-in mouse lines that differ in the integration site of the hCD2-LCR within the mCD8 gene complex were generated, and the influence on expression of the CD8 coreceptor was assessed. In these mice the normal developmental silencing of the CD8 genes in the CD4 lineage is deregulated, and the mice develop CD4(+) cells that also express the CD8 genes. This is accompanied by the physical maintenance of the CD8 genes within an extended loop away from their subchromosomal territory. Further analysis of these mice revealed unexpected fluid chromatin dynamics, whereby the LCR can be initially dominant over the endogenous CD8 gene-repressive regulatory processes present in CD4(+) cells but is continuously contested by them, resulting in the eventual inactivation of the inserted LCR, probably as a result of multiple rounds of replication.
Full-text Article · Sep 2010 · Proceedings of the National Academy of Sciences