Nuclease Hypersensitive Sites Research Articles

Potential G‐quadruplexes within the Promoter Nuclease Hypersensitive Sites of the Heat‐Responsive Genes in Rice

G-quadruplexes (G4s) have been shown to be involved in the regulation of multiple cellular processes. Exploring putative G4-forming sequences (PQSs) in heat-responsive genes of rice and their folding structures under different conditions will help to understand the mechanism in response to heat stress. In this work, we discovered a prevalence of PQSs in nuclease hypersensitive sites within the promoters of heat-responsive genes. Moreover, 50 % of the searched G3 PQSs ((G3+ L1-7 )3+ G3+ ) locate in heat shock transcription factors. Circular dichroism spectroscopy, thermal difference spectroscopy, and UV melting analysis demonstrated the representative PQSs could adopt stable G4s at physiological temperature and potassium concentration. These PQSs were able to stall Klenow fragment (KF) DNA polymerase by the formation of G4s. However, the G4s with Tm values around 50-60 °C could be increasingly unwound by KF with the increase of temperatures from 25 to 50 °C, implying that these G4s could sense the changes in temperature by structural switch. This work offers fresh clues to understanding the potential of G4-involved functions of PQSs and the molecular events in plants in response to heat stress.

The bithorax complex iab-7 Polycomb response element has a novel role in the functioning of the Fab-7 chromatin boundary.

Expression of the three bithorax complex homeotic genes is orchestrated by nine parasegment-specific regulatory domains. Autonomy of each domain is conferred by boundary elements (insulators). Here, we have used an in situ replacement strategy to reanalyze the sequences required for the functioning of one of the best-characterized fly boundaries, Fab-7. It was initially identified by a deletion, Fab-71, that transformed parasegment (PS) 11 into a duplicate copy of PS12. Fab-71 deleted four nuclease hypersensitive sites, HS*, HS1, HS2, and HS3, located between the iab-6 and iab-7 regulatory domains. Transgenic and P-element excision experiments mapped the boundary to HS*+HS1+HS2, while HS3 was shown to be the iab-7 Polycomb response element (PRE). Recent replacement experiments showed that HS1 is both necessary and sufficient for boundary activity when HS3 is also present in the replacement construct. Surprisingly, while HS1+HS3 combination has full boundary activity, we discovered that HS1 alone has only minimal function. Moreover, when combined with HS3, only the distal half of HS1, dHS1, is needed. A ~1,000 kD multiprotein complex containing the GAF protein, called the LBC, binds to the dHS1 sequence and we show that mutations in dHS1, that disrupt LBC binding in nuclear extracts, eliminate boundary activity and GAF binding in vivo. HS3 has binding sites for GAF and Pho proteins that are required for PRE silencing. In contrast, HS3 boundary activity only requires the GAF binding sites. LBC binding with HS3 in nuclear extracts, and GAF association in vivo, depend upon the HS3 GAF sites, but not the Pho sites. Consistent with a role for the LBC in HS3 boundary activity, the boundary function of the dHS1+HS3mPho combination is lost when the flies are heterozygous for a mutation in the GAF gene. Taken together, these results reveal a novel function for the iab-7 PREs in chromosome architecture.

Differentially Regulated Orthologs in Sorghum and the Subgenomes of Maize.

Identifying interspecies changes in gene regulation, one of the two primary sources of phenotypic variation, is challenging on a genome-wide scale. The use of paired time-course data on cold-responsive gene expression in maize (Zea mays) and sorghum (Sorghum bicolor) allowed us to identify differentially regulated orthologs. While the majority of cold-responsive transcriptional regulation of conserved gene pairs is species specific, the initial transcriptional responses to cold appear to be more conserved than later responses. In maize, the promoters of genes with conserved transcriptional responses to cold tend to contain more micrococcal nuclease hypersensitive sites in their promoters, a proxy for open chromatin. Genes with conserved patterns of transcriptional regulation between the two species show lower ratios of nonsynonymous to synonymous substitutions. Genes involved in lipid metabolism, known to be involved in cold acclimation, tended to show consistent regulation in both species. Genes with species-specific cold responses did not cluster in particular pathways nor were they enriched in particular functional categories. We propose that cold-responsive transcriptional regulation in individual species may not be a reliable marker for function, while a core set of genes involved in perceiving and responding to cold stress are subject to functionally constrained cold-responsive regulation across the grass tribe Andropogoneae.

Insight into the Complexity of the i-Motif and G-Quadruplex DNA Structures Formed in the KRAS Promoter and Subsequent Drug-Induced Gene Repression.

Activating KRAS mutations frequently occur in pancreatic, colorectal, and lung adenocarcinomas. While many attempts have been made to target oncogenic KRAS, no clinically useful therapies currently exist. Most efforts to target KRAS have focused on inhibiting the mutant protein; a less explored approach involves targeting KRAS at the transcriptional level. The promoter element of the KRAS gene contains a GC-rich nuclease hypersensitive site with three potential DNA secondary structure-forming regions. These are referred to as the Near-, Mid-, and Far-regions, on the basis of their proximity to the transcription start site. As a result of transcription-induced negative superhelicity, these regions can open up to form unique DNA secondary structures: G-quadruplexes on the G-rich strand and i-motifs on the C-rich strand. While the G-quadruplexes have been well characterized, the i-motifs have not been investigated as thoroughly. Here we show that the i-motif that forms in the C-rich Mid-region is the most stable and exists in a dynamic equilibrium with a hybrid i-motif/hairpin species and an unfolded hairpin species. The transcription factor heterogeneous nuclear ribonucleoprotein K (hnRNP K) was found to bind selectively to the i-motif species and to positively modulate KRAS transcription. Additionally, we identified a benzophenanthridine alkaloid that dissipates the hairpin species and destabilizes the interaction of hnRNP K with the Mid-region i-motif. This same compound stabilizes the three existing KRAS G-quadruplexes. The combined effect of the compound on the Mid-region i-motif and the G-quadruplexes leads to downregulation of KRAS gene expression. This dual i-motif/G-quadruplex-interactive compound presents a new mechanism to modulate gene expression.

Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex.

Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

In Cellulo DNA Analysis: LMPCR Footprinting.

The in cellulo analysis of protein-DNA interactions and chromatin structure is very important to better understand the mechanisms involved in the regulation of gene expression. The nuclease-hypersensitive sites and sequences bound by transcription factors often correspond to genetic regulatory elements. Using the ligation-mediated polymerase chain reaction (LMPCR) technology, it is possible to precisely analyze these DNA sequences to demonstrate the existence of DNA-protein interactions or unusual DNA structures directly in living cells. Indeed, the ideal chromatin substrate is, of course, found inside intact cells. LMPCR, a genomic sequencing technique that map DNA single-strand breaks at the sequence level of resolution, is the method of choice for in cellulo footprinting and DNA structure studies because it can be used to investigate complex animal genomes, including human. The detailed conventional and automated LMPCR protocols are presented in this chapter.

Epigenetic Control of Cell Cycle-Dependent Histone Gene Expression Is a Principal Component of the Abbreviated Pluripotent Cell Cycle

Self-renewal of human pluripotent embryonic stem cells proceeds via an abbreviated cell cycle with a shortened G(1) phase. We examined which genes are modulated in this abbreviated period and the epigenetic mechanisms that control their expression. Accelerated upregulation of genes encoding histone proteins that support DNA replication is the most prominent gene regulatory program at the G(1)/S-phase transition in pluripotent cells. Expedited expression of histone genes is mediated by a unique chromatin architecture reflected by major nuclease hypersensitive sites, atypical distribution of epigenetic histone marks, and a region devoid of histone octamers. We observed remarkable differences in chromatin structure--hypersensitivity and histone protein modifications--between human embryonic stem (hES) and normal diploid cells. Cell cycle-dependent transcription factor binding permits dynamic three-dimensional interactions between transcript initiating and processing factors at 5' and 3' regions of the gene. Thus, progression through the abbreviated G(1) phase involves cell cycle stage-specific chromatin-remodeling events and rapid assembly of subnuclear microenvironments that activate histone gene transcription to promote nucleosomal packaging of newly replicated DNA during stem cell renewal.

Capture and identification of proteins that bind to a GGA-rich sequence from the ERBB2 gene promoter region

The ERBB2 gene (HER2/neu) is overexpressed in many human breast cancers. It is an important therapeutic target and its product protein is a key biomarker for breast cancer. A 28-bp GGA repeat sequence (Pu28-mer) in the nuclease hypersensitive site of the ERBB2 promoter region may play an important role in the regulation of ERBB2 transcription, possibly involving the formation of a G-quadruplex. In order to investigate this possibility, an affinity MALDI-MS approach was used for in vitro protein capture from nuclear extracts from cultured MCF-7 and BT-474 cancer cells at Pu28-mer and control oligonucleotide-modified surfaces. Captured proteins from MCF-7 cells were analyzed by LC-MS/MS. Based on these results, Western blot was then used to interrogate captured proteins from both MCF-7 and the Her-2/neu-positive BT-474 cells. Results support the formation of a G-quadruplex by Pu28-mer, indicated by circular dichroism spectroscopy, that selectively captures transcription factors including Ku70, Ku80, PURA, nucleolin, and hnRNP K. Chromatin immunoprecipitation confirmed binding of Ku70, Ku80, PURA, and nucleolin to ERBB2 promoter in the live BT-474 cells. These findings may lead to a better understanding of the role of non-duplex DNA structures in gene regulation and provide a more complete picture of the regulation of ErbB2 expression in breast cancer. The results also provide a blueprint for development of "genome-inspired" aptamers based on the Pu28-mer sequence for in vitro and in vivo detection of proteins related to regulation of ERBB2 gene expression and breast cancer.

Nucleosome exclusion from the interspecies-conserved central AT-rich region of the Ars insulator

The Ars insulator is a boundary element identified in the upstream region of the arylsulfatase (HpArs) gene in the sea urchin, Hemicentrotus pulcherrimus, and possesses the ability to both block enhancer-promoter communications and protect transgenes from silent chromatin. To understand the molecular mechanism of the Ars insulator, we investigated the correlation between chromatin structure, DNA structure and insulator activity. Nuclease digestion of nuclei isolated from sea urchin embryos revealed the presence of a nuclease-hypersensitive site within the Ars insulator. Analysis of micrococcal nuclease-sensitive sites in the Ars insulator, reconstituted with nucleosomes, showed the exclusion of nucleosomes from the central AT-rich region. Furthermore, the central AT-rich region in naked DNA was sensitive to nucleotide base modification by diethylpyrocarbonate (DEPC). These observations suggest that non-B-DNA structures in the central AT-rich region may inhibit nucleosomal formation, which leads to nuclease hypersensitivity. Furthermore, comparison of nucleotide sequences between the HpArs gene and its ortholog in Strongylocentrotus purpuratus revealed that the central AT-rich region of the Ars insulator is conserved, and this conserved region showed significant enhancer blocking activity. These results suggest that the central AT-rich nucleosome-free region plays an important role in the function of the Ars insulator.

Chromatin disruption in the promoter of Bovine Leukemia Virus during transcriptional activation

Bovine leukemia virus expression relies on its chromatin organization after integration into the host cell genome. Proviral latency, which results from transcriptional repression in vivo, represents a viral strategy to escape the host immune system and likely allows for tumor progression. Here, we discriminated two types of latency: an easily reactivable latent state of the YR2 provirus and a ‘locked’ latent state of the L267 provirus. The defective YR2 provirus was characterized by the presence of nuclease hypersensitive sites at the U3/R junction and in the R/U5 region of the 5′-long terminal repeat (5′-LTR), whereas the L267 provirus displayed a closed chromatin configuration at the U3/R junction. Reactivation of viral expression in YR2 cells by the phorbol 12-myristate 13-acetate (PMA) plus ionomycin combination was accompanied by a rapid but transient chromatin remodeling in the 5′-LTR, leading to an increased PU.1 and USF-1/USF-2 recruitment in vivo sustained by PMA/ionomycin-mediated USF phosphorylation. In contrast, viral expression was not reactivated by PMA/ionomycin in L267 cells, because the 5′-LTR U3/R region remained inaccessible to nucleases and hypermethylated at CpG dinucleotides. Remarkably, we elucidated the BLV 5′-LTR chromatin organization in PBMCs isolated from BLV-infected cows, thereby depicting the virus hiding in vivo in its natural host.

Bone Morphogenetic Protein-2 induces chromatin remodeling and modification at the proximal promoter of Sox9 gene

Sox9 is a key transcription factor which plays an important role in chondrogenesis. Although Bone Morphogenetic Protein-2 (BMP-2) has been reported to induce Sox9 expression, the underlying molecular mechanism remains elusive. Here, we used in vivo approaches to characterize BMP-2-induced alterations in chromatin organization around the Sox9 core promoter. Nuclease hypersensitive site mapping following BMP-2 stimulation showed an inducible hypersensitive site in the Sox9 proximal promoter. Immunoprecipitation (IP) experiments demonstrated that BMP-2 increased the association of the transcription factor NF-Y with histone acetyltransferase p300/CBP. Chromatin immunoprecipitation (ChIP) analysis showed the binding of the NF-Y–p300 complex to the Sox9 gene proximal promoter along with PCAF and RNA polymerase II. We also found that BMP-2 stimulation caused histone hyperacetylation and methylation at the Sox9 gene. Collectively, these data suggest that the activation of Sox9 gene transcription by BMP-2 is associated with chromatin remodeling and histone modification.

GATA-1 Modulates the Chromatin Structure and Activity of the Chicken α-Globin 3′ Enhancer

Long-distance regulatory elements and local chromatin structure are critical for proper regulation of gene expression. Here we characterize the chromatin conformation of the chicken alpha-globin silencer-enhancer elements located 3' of the domain. We found a characteristic and erythrocyte-specific structure between the previously defined silencer and the enhancer, defined by two nuclease hypersensitive sites, which appear when the enhancer is active during erythroid differentiation. Fine mapping of these sites demonstrates the absence of a positioned nucleosome and the association of GATA-1. Functional analyses of episomal vectors, as well as stably integrated constructs, revealed that GATA-1 plays a major role in defining both the chromatin structure and the enhancer activity. We detected a progressive enrichment of histone acetylation on critical enhancer nuclear factor binding sites, in correlation with the formation of an apparent nucleosome-free region. On the basis of these results, we propose that the local chromatin structure of the chicken alpha-globin enhancer plays a central role in its capacity to differentially regulate alpha-globin gene expression during erythroid differentiation and development.

A model for triple helix formation on human telomerase reverse transcriptase (hTERT) promoter and stabilization by specific interactions with the water soluble perylene derivative, DAPER

The promoter of human telomerase reverse transcriptase (hTERT) gene, in the region from − 1000 to + 1, contains two homopurine–homopyrimidine sequences (− 835/− 814 and − 108/− 90), that can be considered as potential targets to triple helix forming oligonucleotides (TFOs) for applying antigene strategy. We have chosen the sequence (− 108/− 90) on the basis of its unfavorable chromatin organization, evaluated by theoretical nucleosome positioning and nuclease hypersensitive sites mapping. On this sequence, anti-parallel triplex with satisfactory thermodynamic stability is formed by two TFOs, having different lengths. Triplex stability is significantly increased by specific interactions with the perylene derivative N,N′-bis[3,3′-(dimethylamino) propylamine]-3,4,9,10-perylenetetracarboxylic diimide (DAPER). Since DAPER is a symmetric molecule, the induced Circular Dichroism (CD) spectra in the range 400–600 nm allows us to obtain information on drug binding to triplex and duplex DNA. The drug-induced ellipticity is significantly higher in the case of triplex with respect to duplex and, surprisingly, it increases at decreasing of DNA. A model is proposed where self-stacked DAPER binds to triplex or to duplex narrow grooves.

Histone Replacement Marks the Boundaries of cis-Regulatory Domains

Cellular memory is maintained at homeotic genes by cis-regulatory elements whose mechanism of action is unknown. We have examined chromatin at Drosophila homeotic gene clusters by measuring, at high resolution, levels of histone replacement and nucleosome occupancy. Homeotic gene clusters display conspicuous peaks of histone replacement at boundaries of cis-regulatory domains superimposed over broad regions of low replacement. Peaks of histone replacement closely correspond to nuclease-hypersensitive sites, binding sites for Polycomb and trithorax group proteins, and sites of nucleosome depletion. Our results suggest the existence of a continuous process that disrupts nucleosomes and maintains accessibility of cis-regulatory elements.

G-quadruplexes in promoters throughout the human genome

Certain G-rich DNA sequences readily form four-stranded structures called G-quadruplexes. These sequence motifs are located in telomeres as a repeated unit, and elsewhere in the genome, where their function is currently unknown. It has been proposed that G-quadruplexes may be directly involved in gene regulation at the level of transcription. In support of this hypothesis, we show that the promoter regions (1 kb upstream of the transcription start site TSS) of genes are significantly enriched in quadruplex motifs relative to the rest of the genome, with >40% of human gene promoters containing one or more quadruplex motif. Furthermore, these promoter quadruplexes strongly associate with nuclease hypersensitive sites identified throughout the genome via biochemical measurement. Regions of the human genome that are both nuclease hypersensitive and within promoters show a remarkable (230-fold) enrichment of quadruplex elements, compared to the rest of the genome. These quadruplex motifs identified in promoter regions also show an interesting structural bias towards more stable forms. These observations support the proposal that promoter G-quadruplexes are directly involved in the regulation of gene expression.

Transcription factor binding aites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity

We have previously identified in the pol gene of HIV-1 a new positive transcriptional regulatory element associated with a nuclease-hypersensitive site (HS7) and containing recognition sites for nuclear proteins [1]. We have next further physically characterized each binding site and have shown that the transcription factors Oct-1, Oct-2, PU.1, Sp1 and Sp3 interact in vitro with the pol region. Chromatin immunoprecipitation assays using HIV-infected cell lines demonstrated that Sp1, Sp3, Oct1 and PU.1 are recruited to the HS7 region in vivo. For each site, we have identified mutations abolishing factor binding to their cognate DNA sequences without altering the underlying amino acid sequence of the integrase. By transient transfection assays, we have demonstrated the involvement of the pol binding sites in the transcriptional enhancing activity of the intragenic region. Our functional results with multimerized wild-type and mutated pol binding sites separately have demonstrated that the PU.1, Sp1, Sp3 and Oct-1 transcription factors regulate the transcriptional activity of a heterologous promoter through their respective HS7 binding sites. Finally, we have investigated the physiological role of the HS7 binding sites in HIV-1 replication and have shown that these sites are important for viral infectivity [2]. Current studies are examining the role of AP-1 binding sites located upstream of the HS7 region in the enhancer activity and in the viral replication cycle.

Chromatin Remodeling and Transcriptional Activity of the Bone-specific Osteocalcin Gene Require CCAAT/Enhancer-binding Protein β-dependent Recruitment of SWI/SNF Activity

Tissue-specific activation of the osteocalcin (OC) gene is associated with changes in chromatin structure at the promoter region. Two nuclease-hypersensitive sites span the key regulatory elements that control basal tissue-specific and vitamin D3-enhanced OC gene transcription. To gain understanding of the molecular mechanisms involved in chromatin remodeling of the OC gene, we have examined the requirement for SWI/SNF activity. We inducibly expressed an ATPase-defective BRG1 catalytic subunit that forms inactive SWI/SNF complexes that bind to the OC promoter. This interaction results in inhibition of both basal and vitamin D3-enhanced OC gene transcription and a marked decrease in nuclease hypersensitivity. We find that SWI/SNF is recruited to the OC promoter via the transcription factor CCAAT/enhancer-binding protein beta, which together with Runx2 forms a stable complex to facilitate RNA polymerase II binding and activation of OC gene transcription. Together, our results indicate that the SWI/SNF complex is a key regulator of the chromatin-remodeling events that promote tissue-specific transcription in osteoblasts.

The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites

In the work reported here we have analyzed the role of the GAGA factor [encoded by the Trithorax-like (Trl) gene] in the enhancer-blocking activity of Frontabdominal-7 (Fab-7), a domain boundary element from the Drosophila melanogaster bithorax complex (BX-C). One of the three nuclease hypersensitive sites in the Fab-7 boundary, HS1, contains multiple consensus-binding sequences for the GAGA factor, a protein known to be involved in the formation and/or maintenance of nucleosome-free regions of chromatin. GAGA protein has been shown to localize to the Fab-7 boundary in vivo, and we show that it recognizes sequences from HS1 in vitro. Using two different transgene assays we demonstrate that GAGA-factor-binding sites are necessary but not sufficient for full Fab-7 enhancer-blocking activity. We show that distinct GAGA sites are required for different enhancer-blocking activities at different stages of development. We also show that the enhancer-blocking activity of the endogenous Fab-7 boundary is sensitive to mutations in the gene encoding the GAGA factor Trithorax-like.

Evidence for nucleosome depletion at active regulatory regions genome-wide.

The identification of nuclease-hypersensitive sites in an active globin gene and in the 5' regions of fruit fly heat shock genes first suggested that chromatin changes accompany gene regulation in vivo. Here we present evidence that the basic repeating units of eukaryotic chromatin, nucleosomes, are depleted from active regulatory elements throughout the Saccharomyces cerevisiae genome in vivo. We found that during rapid mitotic growth, the level of nucleosome occupancy is inversely proportional to the transcriptional initiation rate at the promoter. We also observed a partial loss of histone H3 and H4 tetramers from the coding regions of the most heavily transcribed genes. Alterations in the global transcriptional program caused by heat shock or a change in carbon source resulted in an increased nucleosome occupancy at repressed promoters, and a decreased nucleosome occupancy at promoters that became active. Nuclease-hypersensitive sites occur in species from yeast to humans and result from chromatin perturbation. Given the conservation of sequence and function among components of both chromatin and the transcriptional machinery, nucleosome depletion at promoters may be a fundamental feature of eukaryotic transcriptional regulation.

Progesterone Receptor Deficient in Chromatin Binding Has an Altered Cellular State

Our previous work has shown that the progesterone receptor (PR) can exist in two distinct functional states in mammary adenocarcinoma cells. The differences in function included the ability to activate a promoter in organized chromatin, sensitivity to ligand, and ligand-independent activation. To determine whether these functional differences were because of altered cellular processing, we carried out biochemical analyses of the functionally distinct PRs. Although the majority of PR is localized to the nucleus, biochemical partitioning resulted in a loosely bound (cytosolic) fraction, and a tightly bound (nuclear) fraction. In the absence of progestins, the functionally distinct PRs differed significantly in partitioning between the two fractions. To characterize these fractions further, we analyzed interactions of unliganded PR with chaperones by coimmunoprecipitation. We determined that PR in the cytosolic fraction associated with hsp90 and p23. In contrast, PR in the nuclear fraction consisted of complexes containing hsp90, p23, and FKBP51 as well as PR that was dimerized and highly phosphorylated. Hormone treatment significantly reduced the formation of all PR-chaperone complexes. The hsp90 inhibitor, geldanamycin, similarly blocked transcriptional activity of both functionally distinct receptors. However, the two forms of the PR differed in their ability to associate with the mouse mammary tumor virus promoter in organized chromatin. These findings provide new information about the composition and distribution of mature progesterone receptor complexes in mammary adenocarcinoma cells, and suggest that differences in receptor subcellular distribution have a significant impact on their function. These findings also reveal that transiently expressed steroid receptors may not always be processed like their endogenous counterparts.