A Distant Upstream Locus Control Region Is Critical for Expression of the Kit Receptor Gene in Mast Cells

Abstract

The Kit receptor tyrosine kinase functions in hematopoiesis, melanogenesis, and gametogenesis and in interstitial cells of Cajal. We previously identified two upstream hypersensitive site (HS) clusters in mast cells and melanocytes. Here we investigated the roles of these 5 HS sequences in Kit expression using transgenic mice carrying Kit-GFP reporter constructs. In these mice there is close correspondence between Kit-GFP reporter and endogenous Kit gene expression in most tissues analyzed. Deletion analysis defined the 5 upstream HS cluster region as critical for Kit expression in mast cells. Furthermore, chromatin immunoprecipitation analysis in mast cells showed that H3 and H4 histone hyperacetylation and RNA polymerase II recruitment within the Kit promoter and in the 5 HS region were associated with Kit expression. Therefore, the 5 upstream hypersensitivity sites appear to be critical components of locus control region-mediated Kit gene activation in mast cells.Differential control of gene expression during embryonic development and in the adult organism is mediated by the interaction of the transcription machinery with cis regulatory elements located at the promoter, upstream, or in intronic sequences of a gene. The mechanism by which tissue-specific gene expression is achieved involves the action of transcription factors together with changes in chromatin structure. Chromatin structure is a major determinant of gene expression, and mechanisms of chromatin remodeling are critical components of its regulation. Whereas SWI/SNF-like chromatin-remodeling ATPases disrupt histone-DNA interactions, covalent Nterminal histone modifications, including acetylation, methylation, and phosphorylation, also have important roles in the remodeling of chromatin structure and the regulation of transcription (36). In this way histone modification patterns have been proposed to constitute a histone "code" which specifies downstream functions (45). Hyperacetylation and hypoacetylation of histones H3 and H4 correlate with open/accessible or closed/inaccessible chromatin structures, respectively, and transcriptional activation or repression (22, 31). Thus, the characterization of histone modification patterns has become an important tool in studies of gene expression.The Kit receptor tyrosine kinase (RTK) functions in distinct cell populations during embryonic development and in the postnatal animal (5, 6). During gametogenesis, Kit is expressed in primordial germ cells as they migrate from the allantois to the genital ridge (10, 35). Subsequently, during postnatal gametogenesis, Kit is expressed in spermatogonia and oocytes and in endocrine Leydig and thecal cells (2). In hematopoiesis during embryogenesis and in postnatal animals, Kit is expressed in hematopoietic stem cells and lineage progenitors, as well as in mast cells and eosinophils (5, 18, 48). In melanogenesis, Kit is expressed in migrating melanoblasts during embryonic development, in differentiated melanocytes in hair follicles, and in the gastrointestinal tract in ...

Genetic Inversion in Mast Cell-Deficient Wsh Mice Interrupts Corin and Manifests as Hematopoietic and Cardiac Aberrancy

“…The inversion included a regulatory locus located approximately 150kb upstream of Kit that controls the expression of this receptor in mast cells, and elegant experimental work demonstrated that interference with this regulatory element is the probable cause for mast cell deficiency in the W sh mouse. 16 The current results provide an improved understanding of the W sh inversion. The 3Ј end of the inversion resides 67.5 kb 5Ј to the coding region of Kit in a region devoid of known genes.…”

“…null, the other impaired) at the Kit locus W (white spotting), while the W sh mouse carries an incompletely characterized inversion upstream of Kit that affects a key regulatory element. [11][12][13][14][15][16] Mice with mutations affecting Kit (rather than SCF) are particularly useful because they can be engrafted with cultured mast cells. 17,18 An abnormal phenotype that can be corrected with such engraftment may presumptively be attributed to mast cell deficiency.…”

Mast Cell–deficient KitW-sh “Sash” Mutant Mice Display Aberrant Myelopoiesis Leading to the Accumulation of Splenocytes That Act as Myeloid-Derived Suppressor Cells

“…The 59 breakpoint of this inversion disrupts the Corin gene, leading to cardiac hypertrophy, and the 39 breakpoint is located 72 kbp upstream of the c-Kit transcriptional start site (14,15). Regulatory elements driving the expression of c-Kit in mast cells were mapped within the affected region (16). Thus, it can be assumed that the inversion or additional deletions of cis regulatory elements prevent mast cell-specific c-Kit expression, which ultimately leads to their irreversible demise within a few weeks after birth of sash mice (17,18).…”

Green fluorescent protein transgene driven by Kit regulatory sequences is expressed in hematopoietic stem cells

“…33 These data suggest that upstream sequences are involved in Kit activity, 33 although, unexpectedly, large transgenic constructs including them are very poorly expressed in bone marrow cells. 34 Notably, the upstream sequences are dispensable in our construct. It is possible that these sequences might be involved in a different level of regulation than the promoter and first intron sequences, perhaps controlling chromatin structure and accessibility of the proximal sequences to the transcriptional machinery, rather than directly affecting their activity.…”