ABSTRACT
Special AT-rich binding protein 1 (SATB1) nuclear protein, expressed predominantly in T cells, regulates genes through targeting chromatin remodeling during T-cell maturation. Here we show SATB1 family protein induction during early human adult erythroid progenitor cell differentiation concomitant with epsilon-globin expression. Erythroid differentiation of human erythroleukemia K562 cells by hemin simultaneously increases gamma-globin and down-regulates SATB1 family protein and epsilon-globin gene expression. Chromatin immunoprecipitation using anti-SATB1 anti-body shows selective binding in vivo in the beta-globin cluster to the hypersensitive site 2 (HS2) in the locus control region (LCR) and to the epsilon-globin promoter. SATB1 overexpression increases epsilon-globin and decreases gamma-globin gene expression accompanied by histone hyperacetylation and hypomethylation in chromatin from the epsilon-globin promoter and HS2, and histone hypoacetylation and hypermethylation associated with the gamma-globin promoter. In K562 cells SATB1 family protein forms a complex with CREB-binding protein (CBP) important in transcriptional activation. In cotransfection experiments, increase in epsilon-promoter activity by SATB1 was amplified by CBP and blocked by E1A, a CBP inhibitor. Our results suggest that SATB1 can up-regulate the epsilon-globin gene by interaction with specific sites in the beta-globin cluster and imply that SATB1 family protein expressed in the erythroid progenitor cells may have a role in globin gene expression during early erythroid differentiation.
Subject(s)
Erythroid Cells/cytology , Gene Expression Regulation, Developmental/physiology , Globins/genetics , Matrix Attachment Region Binding Proteins/genetics , Matrix Attachment Region Binding Proteins/metabolism , Cell Differentiation/drug effects , Cell Differentiation/physiology , Chromatin/physiology , Erythroid Cells/drug effects , Hematopoietic Stem Cells/cytology , Hemin/pharmacology , Humans , K562 Cells , Multigene Family , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Transcription, Genetic/physiology , TransfectionABSTRACT
A small library of pyrrole-imidazole polyamide-DNA alkylator (chlorambucil) conjugates was screened for effects on morphology and growth characteristics of a human colon carcinoma cell line, and a compound was identified that causes cells to arrest in the G2/M stage of the cell cycle. Microarray analysis indicates that the histone H4c gene is significantly downregulated by this polyamide. RT-PCR and Western blotting experiments confirm this result, and siRNA to H4c mRNA yields the same cellular response. Strikingly, reduction of H4 protein by >50% does not lead to widespread changes in global gene expression. Sequence-specific alkylation within the coding region of the H4c gene in cell culture was confirmed by LM-PCR. The compound is active in a wide range of cancer cell lines, and treated cells do not form tumors in nude mice. The compound is also active in vivo, blocking tumor growth in mice, without obvious animal toxicity.
Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Chlorambucil/pharmacology , Gene Expression/drug effects , Nylons/pharmacology , Antineoplastic Agents, Alkylating/chemistry , Cell Line, Tumor , Cell Nucleus/chemistry , Cell Nucleus/drug effects , Cell Proliferation/drug effects , Chlorambucil/chemistry , Cross-Linking Reagents/pharmacology , DNA/metabolism , Drug Evaluation, Preclinical , Gene Expression Regulation , Gene Silencing , Gene Targeting , Histones/genetics , Humans , Nylons/chemistryABSTRACT
Pyrrole-imidazole polyamides bind DNA with affinities comparable to those of transcriptional regulatory proteins and inhibit the DNA binding activities of components of the transcription apparatus. If polyamides are to be useful for the regulation of gene expression in cell culture experiments, one pivotal issue is accessibility of specific sites in nuclear chromatin. We first determined the kinetics of uptake and subcellular distribution of polyamides in lymphoid and myeloid cells using fluorescent polyamide-bodipy conjugates and deconvolution microscopy. Then cells were incubated with a polyamide-chlorambucil conjugate, and the sites of specific DNA cleavage in the nuclear chromatin were assayed by ligation-mediated PCR. In addition, DNA microarray analysis revealed that two different polyamides generated distinct transcription profiles. Remarkably, the polyamides affected only a limited number of genes.
Subject(s)
Chromatin/chemistry , Nylons/pharmacology , Transcription, Genetic/drug effects , Alkylation , Apoptosis/drug effects , Binding Sites , Cell Division/drug effects , Cell Line , DNA/chemistry , Gene Expression Profiling , Humans , Lymphocytes/cytology , Lymphocytes/metabolism , Microscopy, Fluorescence , Myeloid Cells/cytology , Myeloid Cells/metabolism , Nylons/chemistry , Nylons/pharmacokinetics , Oligonucleotide Array Sequence AnalysisABSTRACT
We report the cDNA cloning and functional characterization of human cyclin L, a novel cyclin related to the C-type cyclins that are involved in regulation of RNA polymerase II (pol II) transcription. Cyclin L also contains a COOH-terminal dipeptide repeat of alternating arginines and serines, a hallmark of the SR family of splicing factors. We show that recombinant cyclin L interacts with p110 PITSLRE kinase, and that cyclin L antibody co-immunoprecipitates a kinase activity from HeLa nuclear extracts that phosphorylates the carboxyl-terminal domain (CTD) of pol II and splicing factor SC35, and is inhibited by the cdk inhibitor p21. Cyclin L antibody inhibits the second step of RNA splicing in vitro, and recombinant cyclin L protein stimulates splicing under suboptimal conditions. Significantly, the IC(50) for splicing inhibition by p21 is similar to the IC(50) for inhibition of the cyclin L-associated kinase activity. Cyclin L and its associated kinase are thus new members of the pre-mRNA processing machinery.
