Vitamin D binding protein is required to utilize skin-generated vitamin D.

Vitamin D is produced in the skin following exposure to sunlight. Ultraviolet (UV) B (UVB, 280-310 nm) results in isomerization of 7-dehydrocholesterol to previtamin D that spontaneously isomerizes to vitamin D. This pool of skin-derived vitamin D is the major source of vitamin D for animals. However, the mechanisms by which it becomes available remain undefined. It has been assumed that cutaneous vitamin D is transported into the circulation by vitamin D binding protein (DBP), but experimental evidence is lacking. To determine whether cutaneous vitamin D is transported by DBP, we utilized DBP-/- mice that were made vitamin D-deficient. These animals lack measurable 25(OH)D in blood and are hypocalcemic. As controls, DBP+/+ animals were vitamin D depleted and made equally hypocalcemic. UV irradiation of DBP+/+ animals restored serum calcium and serum 25(OH)D while the same treatment of DBP-/- animals failed to show either a serum calcium or 25(OH)D response despite having normal vitamin D production in skin. Intravenous injection of small amounts of recombinant DBP to the vitamin D-deficient DBP-/- mice restored the response to UV light. These results demonstrate a requirement for DBP to utilize cutaneously produced vitamin D.

Long-range looping of a locus control region drives tissue-specific chromatin packing within a multigene cluster.

The relationships of higher order chromatin organization to mammalian gene expression remain incompletely defined. The human Growth Hormone (hGH) multigene cluster contains five gene paralogs. These genes are selectively activated in either the pituitary or the placenta by distinct components of a remote locus control region (LCR). Prior studies have revealed that appropriate activation of the placental genes is dependent not only on the actions of the LCR, but also on the multigene composition of the cluster itself. Here, we demonstrate that the hGH LCR 'loops' over a distance of 28 kb in primary placental nuclei to make specific contacts with the promoters of the two GH genes in the cluster. This long-range interaction sequesters the GH genes from the three hCS genes which co-assemble into a tightly packed 'hCS chromatin hub'. Elimination of the long-range looping, via specific deletion of the placental LCR components, triggers a dramatic disruption of the hCS chromatin hub. These data reveal a higher-order structural pathway by which long-range looping from an LCR impacts on local chromatin architecture that is linked to tissue-specific gene regulation within a multigene cluster.

Functional characterization of a human POU1F1 mutation associated with isolated growth hormone deficiency: a novel etiology for IGHD.

POU1F1, a pituitary-specific POU-homeo domain transcription factor, plays an essential role in the specification of the somatotroph, lactotroph and thyrotroph lineages and in the activation of GH1, PRL and TSHß transcription. Individuals with mutations in POU1F1 present with combined deficiency of GH, PRL and TSH. Here, we identified a heterozygous missense mutation with evidence of pathogenicity, at the POU1F1 locus, in a large family in which an isolated growth hormone deficiency segregates as an autosomal dominant trait. The corresponding p.Pro76Leu mutation maps to a conserved site within the POU1F1 transactivation domain. Bandshift assays revealed that the mutation alters wild-type POU1F1 binding to cognate sites within the hGH-LCR and hGH1 promoter, but not to sites within the PRL promoter, and it selectively increases binding affinity to sites within the hGH-LCR. Co-immunoprecipitation studies reveal that this substitution enhances interactions of POU1F1 with three of its cofactors, PITX1, LHX3a and ELK1, and that residue 76 plays a critical role in these interactions. The insertion of the mutation at the mouse Pou1f1 locus results in a dramatic loss of protein expression despite normal mRNA concentrations. Mice heterozygous for the p.Pro76Leu mutation were phenotypically normal while homozygotes demonstrated a dwarf phenotype. Overall, this study unveils the involvement of POU1F1 in dominantly inherited isolated GH deficiency and demonstrates a significant impact of the Pro76Leu mutation on DNA-binding activities, alterations in transactivating functions and interactions with cofactors. Our data further highlight difficulties in modeling human genetic disorders in the mouse despite apparent conservation of gene expression pathways and physiologic functions.

Autonomous actions of the human growth hormone long-range enhancer.

The human growth hormone (hGH) gene is controlled by a long-range enhancer, HSI, located 14.5 kb 5' to the hGH promoter. HSI establishes a domain of noncoding transcription that is 'looped' to the hGH promoter as an essential step in initiating hGH gene expression. Thus, defining how HSI generates its domain of noncoding transcription is central to understanding its long-range function. Here, we demonstrate that activation of noncoding transcription reflects an HSI-autonomous activity fully independent of interactions with linked gene promoters and occurring in spatial and temporal synchrony with initiation of GH expression in the embryonic pituitary. HSI establishes its noncoding transcription start sites (TSS) over a defined distance from its core determinants and in a manner independent of local primary sequences. The interval between HSI and it TSS co-maps with a domain of disordered and/or highly mobile nucleosomes specific to the pituitary locus. Thus, a localized chromatin reconfiguration by HSI and consequent establishment of an adjacent domain of noncoding transcription constitute initiating events in long-range enhancer function within the hGH locus.

An autoregulatory pathway establishes the definitive chromatin conformation at the pit-1 locus.

The transcription factor Pit-1 (POU1-F1) plays a dominant role in cell lineage expansion and differentiation in the anterior pituitary. Prior studies of the mouse Pit-1 (mPit-1) gene revealed that this master regulatory locus is activated at embryonic day 13.5 (E13.5) by an early enhancer (EE), whereas its subsequent expression throughout adult life is maintained by a more distal definitive enhancer (DE). Here, we demonstrate that the sequential actions of these two enhancers are linked to corresponding shifts in their proximities to the Pit-1 promoter. We further demonstrate that the looping of the definitive enhancer to the mPit-1 promoter is critically dependent on a self-sustaining autoregulatory mechanism mediated by the Pit-1 protein. These Pit-1-dependent actions are accompanied by localized recruitment of CBP and enrichment for H3K27 acetylation within the Pit-1 locus. These data support a model in which the sequential actions of two developmentally activated enhancers are linked to a corresponding shift in higher-order chromatin structures. This shift establishes an autoregulatory circuit that maintains durable expression of Pit-1 throughout adult life.

Tissue specific CTCF occupancy and boundary function at the human growth hormone locus.

The robust and tissue-specific activation of the human growth hormone (hGH) gene cluster in the pituitary and placenta constitutes an informative model for analysis of gene regulation. The five-gene hGH cluster is regulated by two partially overlapping sets of DNase I hypersensitive sites (HSs) that constitute the pituitary (HSI, II, III and V) and placental (HSIII, IV, and V) locus control regions (LCRs). The single placenta-specific LCR component, HSIV, is located at -30 kb to the cluster. Here we generate a series of hGH/BAC transgenes specifically modified to identify structural features of the hGH locus required for its appropriate placental expression. We find that placental specificity is dependent on the overall multigene configuration of the cluster whereas the distance between the cluster and its LCR impacts the level of placental expression. We further observe that a major function of the placental hGH LCR is to insulate the transgene locus from site-of-integration effects. This insulation activity is linked to placenta-specific occupancy of the chromatin architectural protein, CTCF, at HSIV. These data reveal a remarkable combination of structural configurations and regulatory determinants that must work in concert to insure robust and tightly controlled expression from a complex multigene locus.

Neutrophil recruitment to the lung in both C5a- and CXCL1-induced alveolitis is impaired in vitamin D-binding protein-deficient mice.

Knowledge of how neutrophils respond to chemotactic signals in a complex inflammatory environment is not completely understood. Moreover, even less is known about factors in physiological fluids that regulate the activity of chemoattractants. The vitamin D-binding protein (DBP) has been shown to significantly enhance chemotaxis to complement activation peptide C5a using purified proteins in vitro, and by ex vivo depletion of DBP in physiological fluids, but this function has not been determined in vivo. DBP null ((-/-)) mice were used to investigate how a systemic absence of this plasma protein affects leukocyte recruitment in alveolitis models of lung inflammation. DBP(-/-) mice had significantly reduced (~50%) neutrophil recruitment to the lungs compared with their wild-type DBP(+/+) counterparts in three different alveolitis models, two acute and one chronic. The histology of DBP(-/-) mouse lungs also showed significantly less injury than wild-type animals. The chemotactic cofactor function of DBP appears to be selective for neutrophil recruitment, but, in contrast to previous in vitro results, in vivo DBP can enhance the activity of other chemoattractants, including CXCL1. The reduced neutrophil response in DBP(-/-) mice could be rescued to wild-type levels by administering exogenous DBP. Finally, in inflammatory fluids, DBP binds to G-actin released from damaged cells, and this complex may be the active chemotactic cofactor. To our knowledge, results show for the first time that DBP is a significant chemotactic cofactor in vivo and not specific for C5a, suggesting that this ubiquitous plasma protein may have a more significant role in neutrophil recruitment than previously recognized.

Identification of a secondary promoter within the human B cell receptor component gene hCD79b.

The human B cell-specific protein, CD79b (also known as Igß and B29) constitutes an essential signal transduction component of the B cell receptor. Although its function is central to the triggering of B cell terminal differentiation in response to antigen stimulation, the transcriptional determinants that control CD79b gene expression remain poorly defined. In the present study, we explored these determinants using a series of hCD79b transgenic mouse models. Remarkably, we observed that the previously described hCD79b promoter along with its associated enhancer elements and first exon could be deleted without appreciable loss of hCD79b transcriptional activity or tissue specificity. In this deletion setting, a secondary promoter located within exon 2 maintained full levels and specificity of hCD79b transcription. Of note, this secondary promoter was also active, albeit at lower levels, in the wild-type hCD79b locus. The activity of the secondary promoter was dependent on the action(s) of a conserved sequence element mapping to a chromatin DNase I hypersensitive site located within intron 1. mRNA generated from this secondary promoter is predicted to encode an Igß protein lacking a signal sequence and thus unable to serve normal B cell receptor function. Although the physiologic role of the hCD79b secondary promoter and its encoded protein remain unclear, the current data suggest that it has the capacity to play a role in normal as well as pathologic states in B cell proliferation and function.

The role of the serum vitamin D binding protein in the actions of the vitamin D analog eldecalcitol (ED-71) on bone and mineral metabolism.

The vitamin D analog ED-71 (eldecalcitol) has been shown to be superior to calcitriol and its precursor alfacalcidol in maintaining or increasing bone mass in women and animal models with osteoporosis. The mechanism for the greater effectiveness of ED-71 is unknown. In the present study, we tested the hypothesis that the higher activity of ED-71 is due to its higher affinity for the serum vitamin D binding protein (DBP) by comparing the activities of orally administered ED-71, calcitriol and 22-oxacalcitriol (OCT) in wild type (WT) and DBP-ablated (DBPko) mice. In 8-week-old male WT mice, the effects of the analogs on serum and urinary calcium and phosphate were ED-71 > calcitriol > OCT. The results in DBPko mice were identical to those of the WT mice for all parameters tested. In ovariectomized mice, ED-71 was more effective than calcitriol in increasing bone mineral density, but again, there were no differences in the responses of the WT versus DBPko OVX mice. This lack of an effect of DBP ablation on the activities of oral ED-71 occurred despite the finding that peak circulating levels of ED-71 were 100 times lower and disappeared quickly in the DBPko mice while the peak levels at 1 h in WT mice were maintained for at least 24 h. These findings indicate that although DBP has a major influence on circulating levels of vitamin D compounds, it is not responsible for the greater efficacy of ED-71 on bone and mineral metabolism.

Distinct chromatin configurations regulate the initiation and the maintenance of hGH gene expression.

For many mammalian genes, initiation of transcription during embryonic development must be subsequently sustained over extensive periods of adult life. It remains unclear whether maintenance of gene expression reflects the same set of pathways as are involved in initial gene activation. The human pituitary growth hormone (hGH-N) locus is activated in the differentiating somatotrope midway through embryogenesis by a multicomponent locus control region (LCR). DNase I-hypersensitive site I (HSI) of the LCR is essential to full developmental activation of the hGH-N locus. Here we demonstrate that conditional deletion of HSI from the active hGH locus in the adult pituitary effectively silences hGH-N expression. Analyses of chromatin structure and locus positioning demonstrate that a specific subset of the HSI functions active in the embryo retain their HSI dependence in the adult pituitary. These functions sustain engagement of the hGH locus with polymerase II (Pol II) factories, histone acetylation at the hGH-N promoter, and looping of the LCR to its target promoter. These data reveal that HSI is essential to both the maintenance and the initiation phases of gene expression. These observations contribute to our mechanistic understanding of how stable patterns of mammalian gene expression are established in a terminally differentiated cell.

Neurobeachin regulates neurotransmitter receptor trafficking to synapses.

The surface density of neurotransmitter receptors at synapses is a key determinant of synaptic efficacy. Synaptic receptor accumulation is regulated by the transport, postsynaptic anchoring, and turnover of receptors, involving multiple trafficking, sorting, motor, and scaffold proteins. We found that neurons lacking the BEACH (beige-Chediak/Higashi) domain protein Neurobeachin (Nbea) had strongly reduced synaptic responses caused by a reduction in surface levels of glutamate and GABA(A) receptors. In the absence of Nbea, immature AMPA receptors accumulated early in the biosynthetic pathway, and mature N-methyl-d-aspartate, kainate, and GABA(A) receptors did not reach the synapse, whereas maturation and surface expression of other membrane proteins, synapse formation, and presynaptic function were unaffected. These data show that Nbea regulates synaptic transmission under basal conditions by targeting neurotransmitter receptors to synapses.

Synapse associated protein 102 (SAP102) binds the C-terminal part of the scaffolding protein neurobeachin.

Neurobeachin (Nbea) is a multidomain scaffold protein abundant in the brain, where it is highly expressed during development. Nbea-null mice have severe defects in neuromuscular synaptic transmission resulting in lethal paralysis of the newborns. Recently, it became clear that Nbea is important also for the functioning of central synapses, where it is suggested to play a role in trafficking membrane proteins to both, the pre- and post-synaptic sites. So far, only few binding partners of Nbea have been found and the precise mechanism of their trafficking remains unclear. Here, we used mass spectrometry to identify SAP102, a MAGUK protein implicated in trafficking of the ionotropic glutamate AMPA- and NMDA-type receptors during synaptogenesis, as a novel Nbea interacting protein in mouse brain. Experiments in heterologous cells confirmed this interaction and revealed that SAP102 binds to the C-terminal part of Nbea that contains the DUF, PH, BEACH and WD40 domains. Furthermore, we discovered that introducing a mutation in Nbea's PH domain, which disrupts its interaction with the BEACH domain, abolishes this binding, thereby creating an excellent starting point to further investigate Nbea-SAP102 function in the central nervous system.

DNase I hypersensitive site II of the human growth hormone locus control region mediates an essential and distinct long-range enhancer function.

Locus control regions (LCRs) comprise sets of DNA elements capable of establishing autonomous chromatin domains that support robust and physiologically appropriate expression of target genes, often working over extensive distances. Human growth hormone (hGH-N) expression in the pituitary is under the regulation of a well characterized LCR containing four DNase I hypersensitive sites (HSs). The two pituitary-specific HS, HSI and HSII, are located 14.5 and 15.5 kb 5' to the hGH-N promoter. HSI is essential for activation of hGH-N during pituitary development and for sustaining robust activity in the adult. To determine whether the closely linked HSII has a role in hGH-N expression, it was deleted from a previously validated hGH/P1 transgene. Analysis of three independent hGH/P1(ΔHSII) transgenic mouse lines revealed that this deletion had no adverse effect on the formation of HSI, yet resulted in a substantial loss (70%) in hGH-N mRNA expression. This loss of expression was accompanied by a corresponding reduction in recruitment of the pituitary-specific transcription factor Pit-1 to the hGH-N promoter and a selective decrease in promoter occupancy of the elongation-linked isoform of RNA polymerase II. Sufficiency of HSI and HSII in LCR activity was explored by establishing two additional sets of mouse transgenic lines in which DNA segments containing these HS were positioned within the λ phage genome. In this "neutral" DNA context, HSII was required for the recruitment of HAT activity. These data establish HSII as a nonredundant component of the hGH LCR essential for establishment of robust levels of hGH-N gene expression.

An RNA-independent linkage of noncoding transcription to long-range enhancer function.

The detection of noncoding transcription at multiple enhancers within the mammalian genome raises critical questions regarding whether and how this activity contributes to enhancer function. Here, using in vivo analysis of a human growth hormone (hGH) transgene locus, we report that activation of a domain of noncoding transcription adjacent to the long-range hGH-N enhancer, HSI, is established by the enhancer independent of any interactions with its target promoter. We further demonstrate that the appearance of this enhancer-linked noncoding transcription is temporally and spatially concordant with induction of hGH-N in the embryonic pituitary. Finally, we show that the level of transcriptional enhancement of hGH-N by HSI is directly related to the intensity of HSI-dependent noncoding transcription and is fully independent of the structure of the locally transcribed RNA. These data extend our understanding of the relationship of long-range enhancer activity to enhancer-dependent noncoding transcription and establish a model that may be of general relevance to additional mammalian loci.

The role of the hGH locus control region in somatotrope restriction of hGH-N gene expression.

Expression of mammalian GH is normally restricted to somatotropes and somatolactotropes (somatotrope lineages) in the anterior pituitary. The basis for this restriction remains incompletely understood. Recent studies indicate that deoxyribonuclease I hypersensitive site I (HSI) of the hGH locus control region, located at -14.5 kb relative to the hGH-N promoter, acts as a potent long-range enhancer of hGH-N transcription. Here we report that HSI is also critical to somatotrope-restriction of hGH-N expression. Loss of HSI activity, either by direct inactivation of HSI or by interference with HSI-dependent downstream events, results in a relaxation of hGH-N cell-type specification with expansion of hGH-N expression to the full spectrum of Pit-1 positive pituitary cell types. These findings expand the defined roles for HSI of the hGH locus control region to include somatotrope lineage restriction as well as transcriptional enhancement of hGH-N gene expression.

Research resource: T-antigen transformation of pituitary cells captures three novel cell lines in the Pit-1 lineage.

We report the establishment of three distinct pituitary-derived murine cell lines generated by targeted T-antigen-induced transformation. The Pit1/0 line expresses pituitary-specific transcription factor-1 (Pit-1) but lacks expression of GH, prolactin (Prl), or TSH, and the Pit1/Prl line is selectively positive for Pit-1 and Prl. The third line, Pit1/Triple, expresses Pit-1 and all three of the Pit-1-dependent hormones: GH, Prl, and TSHß/glycoprotein hormone α-subunit. The three corresponding transformation events appear to have captured pituitary cells representing: 1) an initial step in the Pit-1(+) lineage, 2) a cell line that corresponds to the differentiated lactotrope, and 3) a novel tri-hormone intermediate that may represent a pivotal step in Pit-1(+) cell lineage differentiation. The documented dependence of the tri-hormone expression in the Pit-1/Triple line on Pit-1 activity supports its potential role in the pathway of pituitary cell differentiation. The presence of a 123-kb human transgene encompassing the hGH locus (hGH/bacterial artificial chromosome) in two of these lines, Pit1/0 and Pit1/Prl, further expands their potential utility to the analysis of gene activation within the hGH gene cluster.

The juxtaposition of a promoter with a locus control region transcriptional domain activates gene expression.

Nonlinear chromatin configurations can juxtapose widely separated elements within a genomic locus; however, it remains unclear how these structures are established and contribute to transcriptional control. A 5'-remote locus control region (LCR) regulates the human growth hormone (hGH-N) gene. HSI, a pituitary-specific component of the hGH LCR, establishes a domain of polymerase II (PolII) transcription 5' to hGH-N. Repression of this transcriptional domain by HSI deletion or PolII blockade decreases hGH-N expression. Here, we show that hGH-N activation is accompanied by positioning of the hGH-N promoter to this LCR transcriptional domain. Selectively blocking LCR transcription inhibits the formation of this active 'looped' conformation. Thus, HSI is crucial for establishing a domain of noncoding PolII transcription, and this domain is intimately linked with chromatin organization of the active hGH-N locus. This integration of LCR transcription with chromatin reconfiguration constitutes a robust pathway for long-range gene activation.

Vitamin D-binding protein influences total circulating levels of 1,25-dihydroxyvitamin D3 but does not directly modulate the bioactive levels of the hormone in vivo.

Mice deficient in the expression of vitamin D-binding protein (DBP) are normocalcemic despite undetectable levels of circulating 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. We used this in vivo mouse model together with cells in culture to explore the impact of DBP on the biological activity of 1,25(OH)(2)D(3). Modest changes in the basal expression of genes involved in 1,25(OH)(2)D(3) metabolism and calcium homeostasis were observed in vivo; however, these changes seemed unlikely to explain the normal calcium balance seen in DBP-null mice. Further investigation revealed that despite the reduced blood levels of 1,25(OH)(2)D(3) in these mice, tissue concentrations were equivalent to those measured in wild-type counterparts. Thus, the presence of DBP has limited impact on the extracellular pool of 1,25(OH)(2)D(3) that is biologically active and that accumulates within target tissues. In cell culture, in contrast, the biological activity of 1,25(OH)(2)D(3) is significantly impacted by DBP. Here, although DBP deficiency had no effect on the activation profile itself, the absence of DBP strongly reduced the concentration of exogenous 1,25(OH)(2)D(3) necessary for transactivation. Surprisingly, analogous studies in wild-type and DBP-null mice, wherein we explored the activity of exogenous 1,25(OH)(2)D(3), produced strikingly different results as compared with those in vitro. Here, the carrier protein had virtually no impact on the distribution, uptake, activation profile, or biological potency of the hormone. Collectively, these experiments suggest that whereas DBP is important to total circulating 1,25(OH)(2)D(3) and sequesters extracellular levels of this hormone both in vivo and in vitro, the binding protein does not influence the hormone's biologically active pool.

An intronic locus control region plays an essential role in the establishment of an autonomous hepatic chromatin domain for the human vitamin D-binding protein gene.

The human vitamin D-binding protein (hDBP) gene exists in a cluster of four liver-expressed genes. A minimal hDBP transgene, containing a defined set of liver-specific DNase I hypersensitive sites (HSs), is robustly expressed in mouse liver in a copy-number-dependent manner. Here we evaluate these HSs for function. Deletion of HSI, located 5' to the promoter (kb -2.1) had no significant effect on hDBP expression. In contrast, deletion of HSIV and HSV from intron 1 repressed hDBP expression and eliminated copy number dependency without a loss of liver specificity. Chromatin immunoprecipitation analysis revealed peaks of histone H3 and H4 acetylation coincident with HSIV in the intact hDBP locus. This region contains a conserved array of binding sites for the liver-enriched transcription factor C/EBP. In vitro studies revealed selective binding of C/EBPalpha to HSIV. In vivo occupancy of C/EBPalpha at HSIV was demonstrated in hepatic chromatin, and depletion of C/EBPalpha in a hepatic cell line decreased hDBP expression. A nonredundant role for C/EBPalpha was confirmed in vivo by demonstrating a reduction of hDBP expression in C/EBPalpha-null mice. Parallel studies revealed in vivo occupancy of the liver-enriched factor HNF1alpha at HSIII (at kb 0.13) within the hDBP promoter. These data demonstrate a critical role for elements within intron 1 in the establishment of an autonomous and productive hDBP chromatin locus and suggest that this function is dependent upon C/EBPalpha. Cooperative interactions between these intronic complexes and liver-restricted complexes within the target promoter are likely to underlie the consistency and liver specificity of the hDBP activation.

Epigenetic activation of the human growth hormone gene cluster during placental cytotrophoblast differentiation.

The hGH cluster contains a single human pituitary growth hormone gene (hGH-N) and four placenta-specific paralogs. Activation of the cluster in both tissues depends on 5' remote regulatory elements. The pituitary-specific locus control elements DNase I-hypersensitive site I (HSI) and HSII, located 14.5 kb 5' of the cluster (position -14.5), establish a continuous domain of histone acetylation that extends to and activates hGH-N in the pituitary gland. In contrast, histone modifications in placental chromatin are restricted to the more 5'-remote HSV-HSIII region (kb -28 to -32) and to the placentally expressed genes in the cluster, with minimal modification between these two regions. These data predict distinct modes of hGH cluster gene activation in the pituitary and placenta. Here we used cell culture models to track structural changes at the hGH locus through placental-gene activation. The data revealed that this process was initiated in primary cytotrophoblasts by histone H3K4 di- and trimethylation and H4 acetylation restricted to HSV and to the individual placental-gene repeat (PGR) units within the cluster. Later stages of transcriptional induction were accompanied by enhancement and extension of these modifications and by robust H3 acetylation at HSV, at HSIII, and throughout the placental-gene regions. These data suggested that elements restricted to HSIII-HSV regions and each individual PGR might be sufficient for activation of the hCS genes. This model was tested by comparing hCS transgene expression in the placentas of mouse embryos carrying a full hGH cluster to that in placentas in which the HSIII-HSV region was directly linked to the individual hCS-A PGR unit. The findings indicate that the HSIII-HSV region and the PGR units, although targeted for initial chromatin structural modifications, are insufficient to activate gene expression and that this process is dependent on additional, as-yet-unidentified chromatin determinants.