Genome-wide Regulatory Roles of the C2H2-type Zinc Finger Protein ZNF764 on the Glucocorticoid Receptor

Abstract

The C2H2-type zinc finger protein ZNF764 acts as an enhancer for several steroid hormone receptors, and haploinsufficiency of this gene may be responsible for tissue resistance to multiple steroid hormones including glucocorticoids observed in a patient with 16p11.2 microdeletion. We examined genome-wide regulatory actions of ZNF764 on the glucocorticoid receptor (GR) in HeLa cells as a model system. ZNF764-and GR-binding sites demonstrated similar distribution in various genomic features. They positioned predominantly around 50-500 kbs from the transcription start sites of their nearby genes, and were closely localized with each other, overlapping in ~37% of them. ZNF764 demonstrated differential on/off effects on GR-binding and subsequent mRNA expression: some genes were highly dependent on the presence/absence of ZNF764, but others were not. Pathway analysis revealed that these 3 gene groups were involved in distinct cellular activities. ZNF764 physically interacted with GR at ligand-binding domain through its KRAB domain, and both its physical interaction to GR and zinc finger domain appear to be required for ZNF764 to regulate GR transcriptional activity. Thus, ZNF764 is a cofactor directing GR transcriptional activity toward specific biologic pathways by changing GR binding and transcriptional activity on the glucocorticoid-responsive genes.Steroid hormones exert diverse physiologic functions and play central roles in human physiology 1,2 . Among them, glucocorticoids are critical for the maintenance of organ homeostasis and adaptive response against circumstance changes 3,4 . On the other hand, sex steroids including androgens are necessary for gender-specific development of reproductive organs in fetal and adolescent phases and acquisition/maintenance of fertility and other functions during reproductive ages [5][6][7][8] . These diverse actions of steroid hormones are mediated by the specific intracellular receptors of the steroid hormone receptor (SR) family, such as the glucocorticoid (GR) and the androgen (AR) receptor 9 . SRs consist of common structural domains, the N-terminal immunogenic (NTD), middle DNA-binding (DBD) and C-terminal ligand-binding (LBD) domain 9 . Their DBD consists of two C4-type zinc fingers with which they physically interact as a dimer with an inverted hexameric palindrome separated by 3 base pairs 9,10 . LBD of these receptors is composed of 12 α -helices and 4 β -sheets, and has a ligand-binding pocket created with its helices 9,11 . These receptors share some DNA-binding motifs, as they maintain high sequence homology in their DBDs due to phylogenic proximity 9 . Ligand-activated and DNA-bound SRs modulate their transcriptional activity by attracting and/or communicating with cofactors, other transcription factors and chromatin-associated molecules through their two transactivation domains, activation function (AF)-1 and -2 9,12 . AF-1 is located in NTD and its activity is ligand-independent, while AF-2 is created on LBD through the ligand-induced conformational change of t...

SIRT1 is a transcriptional enhancer of the glucocorticoid receptor acting independently to its deacetylase activity

“…The human GRα-expressing pRShGRα and its control pRSerbA −1 , and pGR107 that expresses the human GRα under the control of the SP6 promoter were gifts from R. M. Evans (Salk Institute, La Jolla, CA). pGEX-4T3-GRα full-length (FL), NTD, DBD, and LBD, which express corresponding portions of the human GRα fused with GST were previously reported (Nader, Chrousos and Kino, 2009, Fadda, Syed, Mackeh et al, 2017). pEGFP-C1-GRα, which expresses the human GRα N-terminally fused with enhanced green fluorescent protein (EGFP), was also previously reported (Habib, Sadoun, Nader et al, 2017).…”

“…ChIP was performed by using the Pierce Magnetic ChIP Kit (Pierce Inc.) in HeLa cells according to the modified instruction from the manufacturer (Fadda et al, 2017). Briefly, HeLa cells were grown on 10-cm dishes, transfected with control or SIRT1 siRNA, and were treated with or without 10 −6 M of DEX similar to the samples for RNA-seq.…”

C2H2-Type Zinc Finger Proteins: Evolutionarily Old and New Partners of the Nuclear Hormone Receptors

“…128 In a genome-wide binding study using ChIP-sequencing in human HeLa cells, ZNF764- and GR-binding sites are found in close proximity, indicating that ZNF764 modulates GR transcriptional activity by incorporated in the transcriptional complex formed on DNA-bound GR. 129 The exact physiologic functions of ZNF764 in humans are not known, but it appears to regulate the GR activity involved in the stress response organized by the hypothalamic-pituitary-adrenal axis and the AR activity required for fetal development of male-type external genitalia. 128…”

Revealing membrane alteration in cells overexpressing CA IX and EGFR by Surface-Enhanced Raman Scattering

“…Recently, we demonstrated a label-free, SERS based approach for the analysis of erythrocytes membranes 12 , in which unlabelled cells were put in contact with a high uniform and densely packed SERS substrate. Clearly, due to the short-range response of the SERS mechanism 13 , only bio-molecules exposed on the membrane can be efficiently detected, conferring a high-contrast with respect to the bulk Raman contribution 14–16 . It is worth noticing that the spatial uniformity of the plasmonic substrate enhancement factor (EF) 17 is a crucial parameter of such type of investigation, minimising the intrinsic signal fluctuations in SERS imaging 12 .…”