Epsilon-globin Gene Research Articles

Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits.

TRIM28 is a scaffold protein that interacts with DNA-binding proteins and recruits corepressor complexes to cause gene silencing. TRIM28 contributes to physiological functions such as cell growth and differentiation. In the chronic myeloid leukemia cell line K562, we edited TRIM28 using CRISPR/Cas9 technology, and the complete and partial knockout (KO) cell clones were obtained and confirmed using quantitative droplet digital PCR (ddPCR) technology. The amplicon sequencing demonstrated no off-target effects in our gene editing experiments. The TRIM28 KO cells grew slowly and appeared red, seeming to have a tendency towards erythroid differentiation. To understand how TRIM28 controls K562 cell proliferation and differentiation, transcriptome profiling analysis was performed in wild-type and KO cells to identify TRIM28-regulated genes. Some of the RNAs that encode the proteins regulating the cell cycle were increased (such as p21) or decreased (such as cyclin D2) in TRIM28 KO cell clones; a tumor marker, the MAGE (melanoma antigen) family, which is involved in cell proliferation was reduced. Moreover, we found that knockout of TRIM28 can induce miR-874 expression to downregulate MAGEC2 mRNA via post-transcriptional regulation. The embryonic epsilon-globin gene was significantly increased in TRIM28 KO cell clones through the downregulation of transcription repressor SOX6. Taken together, we provide evidence to demonstrate the regulatory network of TRIM28-mediated cell growth and erythroid differentiation in K562 leukemia cells.

Epsilon globin gene expression in developing human fetal tissues.

The discovery of free fetal DNA in plasma of pregnant women has opened a new avenue for non-invasive prenatal diagnosis. We hypothesized that epsilon (ɛ)-globin gene expression could serve as a positive control for the presence of fetal nucleic acid. We measured ɛ-globin mRNA in human fetal tissues and compared concentrations with that measured in adult non-pregnant and pregnant samples. Total RNA was isolated from fetal marrow, liver, blood, and placenta (10-24 weeks gestation), from adult peripheral blood mononuclear cells, and from maternal plasma. RNA was reverse transcribed and quantitative polymerase chain reaction performed for ɛ-globin expression. ɛ-globin gene expression was detected in all fetal samples, was detected in plasma of pregnant women, but was negligible in non-pregnant samples. Relative ɛ-globin gene expression was significantly greater in fetal blood compared to fetal liver, and was minimally expressed in placenta. ɛ-globin gene expression decreased at the highest gestational ages in fetal blood, while expression was greatest at 15-19 weeks in fetal marrow. Fetal ɛ-globin gene expression is significantly greater than adult expression and is increased in maternal plasma compared to non-pregnant samples. ɛ-globin gene expression might serve as a positive control when determining the presence of fetal nucleic acid in total nucleic acid isolated from maternal plasma.

Deciphering the Cis- and Trans-regulatory Roles of KLF6 in Primitive Hematopoiesis

AbstractAbstract 4730Krüppel-like factors (KLFs) are a conserved family of Cys2His2 zinc finger proteins which are important components of eukaryotic cellular transcriptional machinery that controls many biological processes including erythroid differentiation and development. As a transcriptional activator and a tumor suppressor, KLF6 was also involved in hematopoiesis. Klf6−/− mice is embryonic lethal by embryonic day 12.5 and associated with markedly reduced hematopoiesis as well as poorly organized yolk sac vascularization. Moreover, the expression of erythroid differentiation markers including Klf1, Gata1 and Scl are delayed and hematopoietic differentiation is impaired in klf6−/− ES cells. However, the detailed mechanism that KLF6 regulates hematopoiesis is not fully understood.To characterize the role of KLF6 in hematopoiesis, we firstly detected the dynamic expression pattern of KLF6 during erythroid differentiation by mRNA-seq in undifferentiated human embryonic stem cells (hESC), three primary erythroid cells at different developmental stages including ES-derived erythroid cells (ESER), fetal- and adult-type erythroid cells (FLER, PBER). The transcriptome analysis showed that KLF6 expressed at significantly higher level in ESER cells compared with that in other cells. Meanwhile, chromatin immunoprecipitation (ChIP) studies in human K562 cells demonstrated the enrichment of KLF6 on the promoter region of embryonic epsilon-globin gene. These results probably indicate that KLF6 play an important role in primitive hematopoiesis. To clarify whether the erythroid-specific enhancers in the genomic region of KLF6 participate in the regulation of primitive hematopoiesis, we extensively screened the erythroid-specific DNaseI hypersensitive sites (DHSs) in the KLF6 locus, from 70 kb upstream of the transcription start site to 20 kb downstream of the poly(A) site, from DNase-seq data in four erythroid cells including ESER, FLER, PBER, K562 and seven non-erythroid cells. The enhancer activity of these erythroid-specific DHSs was comprehensively characterized by dual-luciferase reporter assay in K562 cells as well as non-erythroid HeLa and HEK293 cells. Three erythroid-specific enhancers located 18–24 kb upstream of human KLF6 were finally characterized, which not only helps to understand the higher expression of KLF6 in ESER, but also hints that KLF6 could participate in primitive hematopoiesis through erythroid-specific enhancers.In conclusion, we depicted the dynamic expression pattern of KLF6 during erythroid differentiation, characterized three erythroid-specific enhancers in KLF6 gene locus, and disclosed the potential role of KLF6 in primitive hematopoiesis. Next, the overexpression and depletion of KLF6 in K562 cells will be executed to further explore whether the abnormal KLF6 will affect the expression and functions of globin genes as well as erythroid-specific transcription factors. Chromosome conformation capture (3C) analysis will be performed to evaluate the interactions between the erythroid-specific enhancers and the cis-regulatory elements of hematopoiesis related genes. Moreover, we will establish morpholino-based klf6 knockdown zebrafish model and study the target genes, interacting networks and pathways in which KLF6 involved. Collectively, these results will address the detailed cis- and trans- regulatory functions and molecular mechanism of KLF6 in regulating hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Developmental niches for embryonic erythroid cells

Primitive erythroid cells (EryP) are the first differentiated cell type to be specified during mammalian embryogenesis. EryP arise from a pool of lineage-restricted progenitors in the yolk sac (YS) and then enter the newly formed embryonic circulation to mature in a stepwise, synchronous fashion. Numbering in the millions in the mid-gestation mouse embryo, EryP are the dominant circulating blood cell prior to the rapid generation of adult-type definitive erythroid (EryD) cells in the fetal liver. The identification of maturational events in this lineage presented a significant challenge, as EryD begin to outnumber EryP in the bloodstream from ∼ E14.5 onwards. We used human epsilon-globin gene regulatory elements to drive lineage-specific expression of a histone-H2B::EGFP fusion protein, allowing us to label the chromatin of EryP during their development and to track and quantify EryP nuclei following their expulsion from the cell. Using this transgenic fluorescent reporter mouse line, we have monitored primitive erythropoiesis in three distinct niches: the YS, where EryP progenitors arise; the circulation, where EryP continue to divide and mature; and the fetal liver, where EryP complete the terminal stages of their differentiation.

Sickle cell disease caused by heterozygosity for Hb S and novel LCR deletion: Report of two patients

The b-globin gene LCR is located approximately 6 kb upstream of the embryonic epsilon-globin gene, and is made up of five DNase I hypersensitive sites (HSs), HS 1-5. LCR plays a pivotal role in regulating the expression of downstream epsilon-, (G)gamma-, (A)gamma-, delta-, and beta-globin genes in cis [1]. Deletions removing the LCR and parts of the downstream beta-globin gene cluster in patients have been described [2]. These individuals present with a (gammadeltabeta)0-thalassemia carrier phenotype. We now report two patients with severe sickle cell disease who were compound heterozygous for Hb S mutation and novel LCR deletion. In one case, HS 1-3 were deleted; in the other, HS 1-5 were deleted. In both cases, the b-like globin genes in cis to the LCR deletions were intact. Genotypically, both patients appeared to have sickle cell trait. Coinherited with either LCR deletion, these individuals presented as sickle cell disease patients. The breakpoints of these LCR deletions were defined. These results affirm that HS 2 and 3 are primarily responsible for conferring erythroid specific high-level expression of cis-linked beta-like globin genes. Furthermore, LCR deletions might cause hemolytic disease of newborns.

All of the human β‐type globin genes compete for LCR enhancer activity in embryonic erythroid cells of yeast artificial chromosome transgenic mice

In primitive erythroid cells of human beta-globin locus transgenic mice (TgM), the locus control region (LCR)-proximal epsilon- and gamma-globin genes are transcribed, whereas the distal delta- and beta-globin genes are silent. It is generally accepted that the beta-globin gene is competitively suppressed by gamma-globin gene expression at this developmental stage. Previously, however, we observed that epsilon-globin gene expression was severely attenuated when its distance from the LCR was extended, implying that beta-globin gene might also be silenced because of its great distance from the LCR. Here, to clarify the beta-globin gene silencing mechanism, we established TgM lines carrying either gamma- or epsilon- plus gamma-globin promoter deletions, without significantly altering the distance between the beta-globin gene and the LCR. Precocious expression of delta- and beta-globin genes was observed in primitive erythroid cells of mutant, but not wild-type TgM, which was most evident when both the epsilon and gamma promoters were deleted. Thus, we clearly demonstrated that the repression of the delta- and beta-globin genes in primitive erythroid cells is dominated by competitive silencing by the epsilon- and gamma-globin gene promoters, and that epsilon- and the other beta-like globin genes might be activated by two distinct mechanisms by the LCR.

The role of transcriptional activator GATA-1 at human -globin HS2

GATA-1 is an erythroid activator that binds β-globin gene promoters and DNase I hypersensitive sites (HSs) of the β-globin locus control region (LCR). We investigated the direct role of GATA-1 interaction at the LCR HS2 enhancer by mutating its binding sites within minichromosomes in erythroid cells. Loss of GATA-1 in HS2 did not compromise interaction of NF-E2, a second activator that binds to HS2, nor was DNase I hypersensitivity at HS2 or the promoter of a linked ε-globin gene altered. Reduction of NF-E2 using RNAi confirmed the overall importance of this activator in establishing LCR HSs. However, recruitment of the histone acetyltransferase CBP and RNA pol II to HS2 was diminished by GATA-1 loss. Transcription of ε-globin was severely compromised with loss of RNA pol II from the transcription start site and reduction of H3 acetylation and H3K4 di- and tri-methylation in coding sequences. In contrast, widespread detection of H3K4 mono-methylation was unaffected by loss of GATA-1 in HS2. These results support the idea that GATA-1 interaction in HS2 has a prominent and direct role in co-activator and pol II recruitment conferring active histone tail modifications and transcription activation to a target gene but that it does not, by itself, play a major role in establishing DNase I hypersensitivity.

A facilitated tracking and transcription mechanism of long-range enhancer function

In the human ε−globin gene locus, the HS2 enhancer in the Locus Control Region regulates transcription of the embryonic ε-globin gene located over 10 kb away. The mechanism of long-range HS2 enhancer function was not fully established. Here we show that the HS2 enhancer complex containing the enhancer DNA together with RNA polymerase II (pol II) and TBP tracks along the intervening DNA, synthesizing short, polyadenylated, intergenic RNAs to ultimately loop with the ε-globin promoter. Guided by this facilitated tracking and transcription mechanism, the HS2 enhancer delivers pol II and TBP to the cis-linked globin promoter to activate mRNA synthesis from the target gene. An insulator inserted in the intervening DNA between the enhancer and the promoter traps the enhancer DNA and the associated pol II and TBP at the insulator site, blocking mid-stream the facilitated tracking and transcription mechanism of the enhancer complex, thereby blocking long-range enhancer function.

First generation microarray-system for identification of primate species subject to bushmeat trade

About a quarter of non-human primate species are threatened by extinction in the near future. Loss of habitat, disease and illegal hunting, especially for the bushmeat trade, are major causes of concern. Here, we develop an identification tool for primate genera using diagnostic nucleotide positions in the epsilon globin gene, apolipoprotein B gene and mitochondrial 12S rRNA. We identified 111 diagnostic nucleotide positions suitable for genotyping by a minisequencing assay in a microarray format. To show the applicability of the microarray, we typed 70 non-human primates representing all primate infraorders. Sixty-five samples were assigned to the correct infraorder, and 32 were assigned to the correct genus (the highest level of taxonomic resolution attempted here). Our results show that it is feasible to distinguish among a high number of primate taxa if the system allows hierarchical assignation of the samples at different taxonomic levels and includes both taxon-specific and redundant positions.

Enhancer Blocking by Chicken β-Globin 5′-HS4: ROLE OF ENHANCER STRENGTH AND INSULATOR NUCLEOSOME DEPLETION

The 5'-HS4 chicken beta-globin insulator functions as a positional enhancer blocker on chromatinized episomes in human cells, blocking the HS2 enhancer of the human beta-globin locus control region from activating a downstream epsilon-globin gene. 5'-HS4 interrupted formation of a domain of histone H3 and H4 acetylation encompassing the 6-kb minilocus and inhibited transfer of RNA polymerase from the enhancer to the gene promoter. We found that the enhancer blocking phenotype was amplified when the insulated locus contained a weakened HS2 enhancer in which clustered point mutations eliminated interaction of the transcription factor GATA-1. The GATA-1 mutation compromised recruitment of histone acetyltransferases and RNA polymerase II to HS2. Enhancer blocking correlated with a significant depletion of nucleosomes in the core region of the insulator as revealed by micrococcal nuclease and DNase I digestion studies. Nucleosome depletion at 5'-HS4 was dependent on interaction of the insulator protein CCCTC-binding factor (CTCF) and was required for enhancer blocking. These findings provide evidence that a domain of active chromatin is formed by spreading from an enhancer to a target gene and can be blocked by a nucleosome-free gap in an insulator.

Mutation screening in the human ɛ-globin gene using single-strand conformation polymorphism analysis

The human epsilon-globin gene is necessary for primitive human erythropoiesis in the yolk sac. Herein we report a non-radioactive single-strand conformation polymorphism (SSCP) approach to screen the human epsilon-globin gene and its regulatory regions for possible mutations and single-nucleotide polymorphisms in normal adult subjects, in order to determine those genomic regions, which are not necessary for its proper regulation and function. We identified no sequence variations apart from the expected 5'epsilon /HincII polymorphism in the fragments analyzed, suggesting that genomic alterations in the epsilon-globin gene are most likely incompatible with normal erythropoiesis and proper embryonic development.

Sequencing and mapping hemoglobin gene clusters in the Australian model dasyurid marsupial Sminthopsis macroura

Comparing globin genes and their flanking sequences across many species has allowed globin gene evolution to be reconstructed in great detail. Marsupial globin sequences have proved to be of exceptional significance. A previous finding of a β(beta)-like ω(omega) gene in the α(alpha) cluster in the tammar wallaby suggested that the α and β cluster evolved via genome duplication and loss rather than tandem duplication. To confirm and extend this important finding we isolated and sequenced BACs containing the α and β loci from the distantly related Australian marsupial Sminthopsis macroura. We report that the α gene lies in the same BAC as the β-like ω gene, implying that the α-ω juxtaposition is likely to be conserved in all marsupials. The LUC7L gene was found 3′ of the S. macroura α locus, a gene order shared with humans but not mouse, chicken or fugu. Sequencing a BAC contig that contained the S. macroura β globin and Ε globin loci showed that the globin cluster is flanked by olfactory genes, demonstrating a gene arrangement conserved for over 180 MY. Analysis of the region 5′ to the S. macroura epsilon (Ε) globin gene revealed a region similar to the eutherian LCR, containing sequences and potential transcription factor binding sites with homology to eutherian hypersensitive sites 1 to 5. FISH mapping of BACs containing S. macroura α and β globin genes located the β globin cluster on chromosome 3q and the α locus close to the centromere on 1q, resolving contradictory map locations obtained by previous radioactive in situ hybridization.

Chromatin structure at the flanking regions of the human beta-globin locus control region DNase I hypersensitive site-2: proposed nucleosome positioning by DNA-binding proteins including GATA-1

The human beta-globin locus control region DNase I hypersensitive site-2 (LCR HS-2) is erythroid-specific and is located 10.9 kb upstream of the epsilon-globin gene. Most studies have only examined the core region of HS-2. However, previous studies in this laboratory indicate that positioned nucleosomes are present at the 5′- and 3′-flanking regions of HS-2. In addition, footprints were observed that indicated the involvement of DNA-binding proteins in positioning the nucleosome cores. A consensus GATA-1 site exists in the region of the 3′-footprint. In this study, using an electrophoretic mobility shift assay (EMSA) and DNase I footprinting, we confirmed that GATA-1 binds in vitro at the 3′-end of HS-2. An additional GATA-1 site was found to bind GATA-1 in vitro at a site positioned 40 bp upstream. At the 5′-end of HS-2, DNase I footprinting revealed a series of footprints showing a marked correlation with the in vivo footprints. EMSA indicated the presence of several erythroid-specific complexes in this region including GATA-1 binding. Sequence alignment for 12 mammalian species in HS-2 confirmed that the highest conservation to be in the HS-2 core. However, a second level of conservation extends from the core to the sites of the proposed positioning proteins at the HS-2 flanking regions, before declining rapidly. This indicates the importance of the HS-2 flanking regions and supports the proposal of nucleosome positioning proteins in these regions.

Genomic and phylogenetic footprinting at the epsilon-globin silencer region in intact human cells

Chromatin structure at the silencer region of the epsilon-globin promoter was investigated using novel nitrogen mustards as probes of protein–DNA interactions. Sites of protection and enhancement that corresponded to known transcription factor binding sites were detected in both K562 and HeLa cells at this gene region. Protection was observed at several sites including the GATA-1/YY1 motifs. Of particular interest was a large 155 bp footprint that was observed at the epsilon-globin gene silencer region of the promoter. This large footprint was consistent with the presence of a positioned nucleosome core in intact human cells at this silencer region. Additionally, the DNA sequence at the epsilon-globin silencer and promoter was compared for 11 mammalian species. Significant areas of conservation were found that correlated with known transcription factor binding motifs. This phylogenetic footprinting analysis was compared to the genomic footprinting data at the epsilon-globin silencer region.

Human beta-globin locus control region HS5 contains CTCF- and developmental stage-dependent enhancer-blocking activity in erythroid cells.

The human beta-globin locus contains five developmentally regulated beta-type globin genes. All five genes depend on the locus control region (LCR), located at the 5' end of the locus, for abundant globin gene transcription. The LCR is composed of five DNase I-hypersensitive sites (HSs), at least a subset of which appear to cooperate to form a holocomplex in activating genes within the locus. We previously tested the requirement for proper LCR polarity by inverting it in human beta-globin yeast artificial chromosome transgenic mice and observed reduced expression of all the beta-type globin genes regardless of developmental stage. This phenotype clearly demonstrated an orientation-dependent activity of the LCR, although the mechanistic basis for the observed activity was obscure. Here, we describe genetic evidence demonstrating that human HS5 includes enhancer-blocking (insulator) activity that is both CTCF and developmental stage dependent. Curiously, we also observed an attenuating activity in HS5 that was specific to the epsilon-globin gene at the primitive stage and was independent of the HS5 CTCF binding site. These observations demonstrate that the phenotype observed in the LCR-inverted locus was in part attributable to placing the HS5 insulator between the LCR HS enhancers (HS1 to HS4) and the promoter of the beta-globin gene.

Evolutionary conservation and functional synergism of curved DNA at the mouse epsilon- and other globin-gene promoters.

Human and mouse globin genes were separated approximately 200 million years ago but still share homology and synergism in many aspects including DNA structure. We first mapped DNA bend sites in the mouse epsilon-globin gene and found that these sites were distributed in a regular manner except in the coding region and their overall average distance was 650.7 bp. The first bend site upstream of the cap site (MepsilonB-1, -334 to -147 bp) was found to contain A + T-rich sequences and features contributing to DNA curvature by computer analysis. Transcription assays using deletion constructs indicated strong promoter activity up to bp -215 in erythriod K562 cells. Therefore, the MepsilonB-1 site was located immediately upstream of the promoter region. A reporter gene assay using a series of constructs containing the promoter region revealed that the MepsilonB-1 site showed repressor activity, and on replacement of the DNA curvature with one from another source the activity was retained. A similar feature was found in the other conserved B-1 sites in the human, bovine, and rabbit beta-like globin genes, with the exception of an unconserved B-1 site in the chicken beta-globin gene. A common feature of these conserved B-1 sites was not the nucleotide sequences but the DNA curvature. Furthermore, a unique nucleosome phase at the MepsilonB-1 site was likely to be directed by DNA curvature. Based on these results, DNA curvature is one of the major features of these promoter regions which might influence transcription through nucleosome positioning.

Beta-globin locus control region HS2 and HS3 interact structurally and functionally.

The overall structure of the DNase I hypersensitive sites (HSs) that comprise the beta-globin locus control region (LCR) is highly conserved among mammals, implying that the HSs have conserved functions. However, it is not well understood how the LCR HSs, either individually or collectively, activate transcription. We analyzed the interactions of HS2, HS3 and HS4 with the human epsilon- and beta-globin genes in chromatinized episomes in fetal/embryonic K562 cells. Only HS2 activates transcription of the epsilon-globin gene, while all three HSs activate the beta-globin gene. HS3 stimulates the beta-globin gene constitutively, but HS2 and HS4 transactivation requires expression of the transcription factor EKLF, which is not present in K562 cells but is required for beta-globin expression in vivo. To begin addressing how the individual HSs may interact with one another in a complex, we linked the beta-globin gene to both the HS2 and HS3. HS2 and HS3 together resulted in synergistic stimulation of beta-globin transcription. Unexpectedly, mutated, inactive forms of HS2 impeded the activation of the beta-globin gene by HS3. Thus, there appear to be distinct interactions among the HSs and between the HSs and the globin genes. These preferential, non-exclusive interactions may underlie an important structural and functional cooperativity among the regulatory sequences of the beta-globin locus in vivo.

Identification of α NF-κB site in the negative regulatory element(ɛ-NRAII) of human ɛ-globin gene and its binding protein NF-κB p50 in the nuclei of K562 cells

The developmental control of the human epsilon-globin gene expression is mediated by transcription regulatory elements in the 5' flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar to NF-kappaB consensus sequence resides in the negative regulatory element (-3028bp approximately -2902bp, termed e-NRAII) 5' to the cap site of this gene. NRF DNA fragment (-3010bp approximately -2986bp) containing the NF-kappaB motif similar sequence was synthesized and used in electrophoresis mobility shift assay (EMSA) and competitive analysis. Data showed that a protein factor from nuclear extracts of K562 cells specifically interacted with NRF DNA fragment. The synthetic NF DNA fragment (containing NF-kappaB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-kappaB motif) could not, suggesting that the binding protein is a member of NF-kappaB/Rel family. Western blot assay demonstrated that the molecular weight of NF-kappaB protein in the nuclei of K562 cells is 50ku. We suggested that NF-kappaB p50 may play an important role in the regulation of human epsilon-globin gene expression.

In vivo DNA-protein interactions at hypersensitive site 3.5 of the human β-globin locus control region

Using ligation-mediated polymerase chain reaction and in vivo footprinting methods to study the status of DNA-protein interactions at hypersensitive site 3.5 (HS3.5) of the locus control region in K562 and HEL cells, we found that there was protein occupancy in vivo at HS3.5 in both cell lines and the status of DNA-protein interaction was different between K562 and HEL. These data provide direct evidence that specific nuclear factor-DNA complexes form in vivo at functionally important sequence motifs of the HS3.5 in erythroid cells. This indicates that HS3.5 may play an important role in the regulation of the beta-globin gene cluster. K562 is a human erythroleukemia cell line in which the embryonic epsilon-globin gene is predominantly expressed, while the HEL cell line expresses predominantly the fetal beta-globin genes. Thus, HS3.5 might also be involved in the regulation of developmental stage-specific expression of beta-globin genes. Our results are also consistent with the model that each hypersensitive site acts as a functional unit and HS3.5 may facilitate the formation of the HS3 functional unit.

The effect of chromatin structure on cisplatin damage in intact human cells.

The influence of chromatin structure on cisplatin DNA damage was investigated in intact human cells. The epsilon-globin gene promoter was utilised as the target DNA sequence and the terminal transferase-dependent PCR technique was employed to examine adduct formation at base pair resolution. It was found that cisplatin preferentially damaged at runs of consecutive guanine bases in intact cells. By comparing the relative intensity of adduct formation in intact cells and in purified genomic DNA, it was possible to assess the influence of chromatin proteins on the extent of cisplatin DNA damage. Enhanced damage in intact cells was found at the CACC site where a member of the Sp1 family of proteins is thought to bind. It is postulated that protein binding at this site bends the DNA double-helix so that enhanced cisplatin binding occurs. The altered DNA binding of cisplatin in the presence of chromatin proteins could be important in the properties of cisplatin as an anti-tumour drug.