View larger version: In this Resource-efficient cooking In a new Functin Download as Thyroid Function Enhancers Slide. The repressive effects Enhsncers be attributed to direct promoter repression. Moreover, B vitamin foods vast majority of the hyperacetylated regions are positioned in introns and intergenic regions S2F Figwhere gene regulatory regions are most frequently positioned [ 37 ]. McInerney EM, Rose DW, Flynn SE, Westin S, Mullen TM, et al. The gene products are involved in neurotransmission, physiology, signalling, transcriptional regulation and cell cycle.

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A GST-pulldown experiments were carried out using the indicated E. coli expressed GST-fusion proteins. B N2a cells were transfected with the indicated episomes and expression vector for full length TRβ or for TRβ DBD and incubated in the absence or presence of T3.

Thus the DBD of TR harbors two potentially important functions for enhancer blocking, such as DNA binding and an interface for the physical interaction with CTCF.

Therefore we functionally tested the TR-DBD for enhancer blocking. We used cells that do not express endogenous TR N2a cells and expressed either full length TR or the DNA binding domain of TR. In contrast to the TR-DBD, only full length TR results in strong enhancer blocking of the F1F2 episome Fig.

This indicates that the TR-DBD is not sufficient for enhancer blocking and that other domains outside the DNA binding domain of TR are needed as well to mediate enhancer blocking of the CTCF bound module and the unliganded TR. CTCF binding sites have been found next to thyroid or steroid receptor binding sites in several cases [8] , [39] , [40].

For a subset of these sites we could demonstrate that enhancer blocking is regulated by thyroid hormone [8]. Therefore, we wanted to know the frequency of combined binding sites for CTCF and the thyroid hormone receptor in the genome.

This ratio is slightly higher than random sequences associated with CTCF bound regions. Clearly, the positive control to search for the CTCF consensus within these regions revealed a significant 22 fold higher frequency as compared to random controls.

We checked the PSSMs for different half-site models against whole genome datasets of the estrogen receptor ER and the retinoic acid receptor alpha RARα [40] , [41] in order to validate our approach in identifying such elements next to CTCF sites.

In these calculations we found that the respective half-site models IR3 and DR5 showed no significant enrichment when we compared the genome-wide binding datasets with control sequences data not shown.

Previous analysis of estrogen receptor binding sites in comparison with CTCF binding revealed that CTCF site distribution strongly correlated with gene density in contrast to ER sites [20]. Based on the CTCF distribution, the genome can be dissected into sequence blocks framed by CTCF sites.

About such CTCF blocks have been found to contain ER binding sites [42]. The authors concluded that CTCF confines the distal action of the estrogen receptor.

This would argue for a clear separation of CTCF and ER sites in function and space for most of the binding sites.

This is in line with the finding that of ER sites analyzed, about colocalized with the CTCF motif [40]. Overall, the ratio of composite CTCF and TR binding sites relative to the total number of TR or CTCF sites is within the same magnitude as found for ER and CTCF.

The importance of low frequency composite elements has been illustrated by the recent finding that binding of Oct4 and CTCF to a composite element of the Xist gene is required for the regulation of X chromosome inactivation [25] Fig. Nevertheless, there is no overall correlation of combined binding of CTCF and Oct4 in the genome [26].

Likewise, we do not observe an enrichment of the Oct4 motif in the vicinity of CTCF binding regions as compared to random genomic regions see Fig. Enhancer blocking elements may require multiple CTCF sites for optimal function, as exemplified at the H19 locus or the Xist and Tsix locus [15] , [50] , [51] top.

Long range chromatin interaction is not only required for enhancer blocking, but for X chromosomal pairing and HLA locus interaction as well. In addition to CTCF, this involves Yy1 and Oct4 [25] , [53] , [62] third row or CIITA at the CTCF site and RFX at the interacting site [63] , [64] bottom row.

Episomal constructs are able to mimic endogenous chromatin and can be used for insulator analysis [43] — [46]. We found that for the episomal vectors that we constructed, the analysis of enhancer blocking is also possible. Addition of T3 relieves enhancer blocking, an important control that demonstrates that the episomal system can be used to study T3 regulated enhancer blocking.

Thereby we identified a new composite element at the ESRRA gene, which indeed mediates T3 regulation of enhancer blocking. Furthermore, CTCF and inhibition of histone deacetylation showed similar effects in this system as has been demonstrated for endogenous genes.

Both the enhancer blocking activity of CTCF as well as the transcriptional repression of rDNA genes by CTCF require poly ADP ribosylation [36] , [47]. Tumor suppressor silencing has been recently shown to be caused by a deficiency in poly ADP ribosylation [37].

In line with these results we could demonstrate that episomal enhancer blocking mediated by a composite binding site is sensitive to a poly ADP ribosylation inhibitor as well. Furthermore, changes in histone acetylation in the context of enhancer blocking or insulation have been shown.

For CTCF binding sites, an increase in acetylation of histone H3 as well as of H4 has been reported [48] , [49]. In contrast, CTCF binding to histone deacetylase activity has been found as well [35] , and functional episomal tests of the β globin insulator demonstrated a general histone deacetylation in the region extending from the enhancer to the gene [43].

Why is the ESRRA gene down-regulated after T3 treatment, whereas the assay shown above indicates a loss of enhancer blocking by T3? We can only speculate that from the different promoters of the gene one may be induced, which may cause other promoters to be turned of. This assay revealed for the first time that in the case of two composite elements a functional synergy between CTCF and the unliganded TR confers enhancer blocking.

This shows that, similar to enhancer elements which are comprised of functional modules binding sites for enhancer factors , enhancer blockers are generated from functional and synergizing modules as well Fig.

Again, in analogy to enhancer elements, functional modules can be multimers of identical factors CTCF as in the case of the H19 locus [50] , [51] or in the X inactivation locus of the active mammalian X chromosome [15] , or different factors such as combinations of CTCF with Oct4 [25] , Kaiso [52] , Yy1 [53] or TR, as shown here.

In these cases Fig. Half site models for nuclear receptor were downloaded from TRANSFAC database. Next we built PSSMs with the appropriate spacing ER6, DR4, DR0, IR4. The PSSM for CTCF was derived from published genomewide CTCF binding data [20] by scanning the top binding regions with the MEME tool [54].

To control for biases in the sequence composition of the CTCF binding regions we shuffled the base positions of the PSSMs and used these to scan the CTCF binding regions. In case of the NR half sites models we tested all possible permutations using the same permutation in both half sites.

Empirical p-values were calculated as the fraction of simulations that produced a number of mapped features as extreme as observed in the real data [56]. A second MCS PstI, EcoRI, BglII, EcoRV, Spe was inserted into the ClaI site of the pGL3-control vector Promega.

pR-E was generated by digesting the pGL3-MCSII vector with BamHI fill in and NheI and cloned in NotI fill in and NheI cut pREP4-ss. pREP4-ss is a pREP4 vector Invitrogen , where the RSV LTR promoter was removed. The F1F2 element of the chicken lysozyme gene was amplified with primer pairs containing SpeI and BglII restriction sites.

The PCR product was cloned into the BglII and SpeI sites of the MCSII of the pGL3-MCSII vector generating pGL3F1F2. pRF1F2E was generated by digesting the pGL3F1F2 vector with BamHI and XhoI, and was cloned blunt end into pREP4-ss NotI blunt ended.

The F1F2 element was multimerized by ligating PCR products amplified from the pGL3F1F2 vector with primers containing SpeI and XbaI restriction sites. pRF1mutF2E was generated as follows: Primer containing the F1mut binding site were annealed and directly cloned into the XbaI, HindIII site of pBK-CMV Stratagene.

Primer containing the F2 element were annealed and directly cloned into the HindIII, BamHI site of pBK-CMV F1mut.

pGL3F1mutF2 was generated by digested pBK-CMVF1mutF2 with XbaI and SpeI and cloning the fragment into the SpeI site of pGL3-MCSII. pRF1mutF2E was generated by digesting pGL3-F1mutF2 with SalI fill in and NheI and cloned into pREP4-ss cut with NotI fill in and NheI.

pRF1F2mutE was cloned in the same manner with F1 and F2mut oligos. pRCOMTE was generated by PCR amplification of the genomic locus with corresponding primer pairs containing SpeI and XbaI restriction sites and subcloned into pBSK Stratagene generating pBSK-COMT. To multimerize binding sites pBSK-COMT was cut with SpeI and NotI and ligated into pBSK-COMT cut with XbaI and NotI.

pBSK-COMT was digested with with XbaI and SpeI and cloned into pGL3-MCSII digested with SpeI to generate pGL3-COMT and again cloned as a BamH1 fill in NheI fragment into pREP4-ss digested with NotI fill in and NheI. All other newly identified CTCF and TR binding sites were cloned in the same manner.

To generate enhancer-less episomes the corresponding pGL3-vector was cut with EcoRV and NheI and cloned into pREP4-ss digested with NotI fill in and NheI.

N2aβ and T cells were transfected using the calcium phosphate method essentially as described [58]. In detail, cells were transfected with 1 µg reporter plasmid per 6-well dish or as indicated. Cells were harvested after 40 h of TSA treatment.

and cells were collected 14 h later. Radiolabeled DNA probes Table S1 were generated by phosphorylation with gamma 32 P ATP and subsequently annealed. The probes were incubated with 0.

Recombinant proteins were prepared as described previously [59]. The binding reaction was performed in PBS [pH 7. For immunoprecipitation we used antibodies specific for CTCF [61] and preimmune serum. Gene specific PCR mixtures contained 1 µl of DNA, 0.

Annealing temperatures and cycling conditions were determined empirically for each primer set. GST and GST fusion proteins were expressed in Escherichia coli BL Bacteria were induced with 0.

Recombinant proteins were purified with glutathione—sepharose beads Amersham Pharmacia Biotech AB and analyzed on SDS—PAGE to normalize protein amounts Fig. After 2 h incubation at 4°C the beads were washed 3 times with 1 ml of binding buffer without BSA. The bound proteins were eluted with SDS sample buffer, fractionated on SDS—PAGE visualized by fluorography.

ChIP-assay demonstrates in vivo binding of CTCF. ChIP was performed using chromatin from HeLa cells and immunoprecipitated using antibodies against CTCF. Specific primers see Table S1 for the CTCF target sites CTS were used in the PCR-reaction.

Negative controls: nonspecific antibody IGG and a nonbinding sequence ESRRα-control. Specificity of in vitro binding of TRβ to predicted target sites. coli expressed GST and GST-TR with the indicated radioactively labeled probe.

For competition experiments a 0. Arrow marks the TR specific shift. Tetracycline inducible shRNA-mediated knock-down of CTCF. Protein levels were measured by western blotting using an anti-CTCF antibody or GAPDH as control. Coomassie stained gel shows expression levels of GST-fusion proteins. Asterisks mark the corresponding GST-fusion proteins.

We would like to thank Helena Klenova for the CTCF antibody and Helmut Dotzlaw for careful reading of the manuscript. Additionally we would like to thank Peter Seum for technical assistance. Conceived and designed the experiments: OW CW MB JL RR.

Performed the experiments: OW CW FAU. Analyzed the data: OW CW MB FAU. Wrote the paper: OW MB RR. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Article Authors Metrics Comments Media Coverage Reader Comments Figures.

Abstract The conserved 11 zinc-finger protein CTCF is involved in several transcriptional mechanisms, including insulation and enhancer blocking. Introduction The conserved 11 zinc-finger protein CTCF is involved in several transcriptional mechanisms, such as gene activation [1] , gene repression [2] , [3] and enhancer blocking [4] — [13].

Results Genome wide search for binding sites for TR and for CTCF In order to analyze whether the occurrence of composite CTCF and TR binding sites is a common feature for many or most CTCF binding sites, or whether this is limited to a subset of CTCF sites, we examined the distribution of CTCF and thyroid hormone receptor binding elements in a genome-wide fashion.

Download: PPT. Figure 2. Schematic representation of genes with a composite element. Composite elements are bound by TR and CTCF In vivo binding of CTCF to the gene regions shown in Figure 2 has been documented by genome wide binding analysis [20].

Figure 3. In vitro binding assays EMSA show direct binding of CTCF and TRβ to predicted target sites. Table 1. Hormone sensitive enhancer blocking on episomes In order to functionally analyze hormone regulated enhancer blocking, we used a test system based on episomal vectors.

Figure 4. F1F2 and ESRRα composite elements mediate hormone sensitive enhancer blocking on episomes. Functional CTS composite elements depend on both, TR and CTCF Since T3 relieves the enhancer blocking activity from the F1F2 and the ESRRA composite elements, we wanted to address a possible contribution of the TR to enhancer blocking in the absence of T3.

Figure 6. TR requires more than just the CTCF interaction domain to mediate enhancer blocking. Discussion CTCF binding sites have been found next to thyroid or steroid receptor binding sites in several cases [8] , [39] , [40].

Materials and Methods Motif scanning Half site models for nuclear receptor were downloaded from TRANSFAC database. Plasmid construction A second MCS PstI, EcoRI, BglII, EcoRV, Spe was inserted into the ClaI site of the pGL3-control vector Promega.

Electrophoretic mobility shift assay Radiolabeled DNA probes Table S1 were generated by phosphorylation with gamma 32 P ATP and subsequently annealed.

GST-pulldown GST and GST fusion proteins were expressed in Escherichia coli BL Supporting Information. Figure S1. s 0. Figure S2. Figure S3. Figure S4. Table S1. List of oligonucleotides used in different applications. Acknowledgments We would like to thank Helena Klenova for the CTCF antibody and Helmut Dotzlaw for careful reading of the manuscript.

Author Contributions Conceived and designed the experiments: OW CW MB JL RR. References 1. Vostrov AA, Quitschke WW The zinc finger protein CTCF binds to the APBbeta domain of the amyloid beta-protein precursor promoter.

Evidence for a role in transcriptional activation. J Biol Chem — View Article Google Scholar 2. Baniahmad A, Steiner C, Kohne AC, Renkawitz R Modular structure of a chicken lysozyme silencer: involvement of an unusual thyroid hormone receptor binding site.

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Cell Mol Life Sci. Bell AC, Felsenfeld G Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Chao W, Huynh KD, Spencer RJ, Davidow LS, Lee JT CTCF, a candidate trans-acting factor for X-inactivation choice. Science — Kanduri C, Fitzpatrick G, Mukhopadhyay R, Kanduri M, Lobanenkov V, et al.

Takai D, Gonzales FA, Tsai YC, Thayer MJ, Jones PA Large scale mapping of methylcytosines in CTCF-binding sites in the human H19 promoter and aberrant hypomethylation in human bladder cancer. Hum Mol Genet — Lefevre P, Witham J, Lacroix CE, Cockerill PN, Bonifer C The LPS-induced transcriptional upregulation of the chicken lysozyme locus involves CTCF eviction and noncoding RNA transcription.

Mol Cell — Cytogenet Genome Res — Kim TH, Abdullaev ZK, Smith AD, Ching KA, Loukinov DI, et al. Desvergne B How do thyroid hormone receptors bind to structurally diverse response elements? Mol Cell Endocrinol — van Helden J Regulatory sequence analysis tools. Nucleic Acids Res — Podvinec M, Kaufmann MR, Handschin C, Meyer UA NUBIScan, an in silico approach for prediction of nuclear receptor response elements.

Mol Endocrinol — Sandelin A, Wasserman WW Prediction of nuclear hormone receptor response elements. Donohoe ME, Silva SS, Pinter SF, Xu N, Lee JT The pluripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting.

Chen X, Xu H, Yuan P, Fang F, Huss M, et al. De K, Ghosh G, Datta M, Konar A, Bandyopadhyay J, et al. J Endocrinol — Feng X, Jiang Y, Meltzer P, Yen PM Transgenic targeting of a dominant negative corepressor to liver blocks basal repression by thyroid hormone receptor and increases cell proliferation.

Flores-Morales A, Gullberg H, Fernandez L, Stahlberg N, Lee NH, et al. Park EA, Jerden DC, Bahouth SW Regulation of phosphoenolpyruvate carboxykinase gene transcription by thyroid hormone involves two distinct binding sites in the promoter.

Biochem J Pt 3 : — Jothi R, Cuddapah S, Barski A, Cui K, Zhao K Genome-wide identification of in vivo protein-DNA binding sites from ChIP-Seq data. Shulemovich K, Dimaculangan DD, Katz D, Lazar MA DNA bending by thyroid hormone receptor: influence of half-site spacing and RXR.

Wendt KS, Yoshida K, Itoh T, Bando M, Koch B, et al. Kanduri M, Kanduri C, Mariano P, Vostrov AA, Quitschke W, et al. Lutz M, Burke LJ, Barreto G, Goeman F, Greb H, et al. Yu W, Ginjala V, Pant V, Chernukhin I, Whitehead J, et al. Witcher M, Emerson BM Epigenetic silencing of the p16 INK4a tumor suppressor is associated with loss of CTCF binding and a chromatin boundary.

Perez-Juste G, Garcia-Silva S, Aranda A An element in the region responsible for premature termination of transcription mediates repression of c-myc gene expression by thyroid hormone in neuroblastoma cells.

Szabo PE, Pfeifer GP, Mann JR Parent-of-origin-specific binding of nuclear hormone receptor complexes in the HIgf2 imprinting control region. Hua S, Kallen CB, Dhar R, Baquero MT, Mason CE, et al. Z associated with breast cancer progression. Mol Syst Biol 4: Hua S, Kittler R, White KP Genomic antagonism between retinoic acid and estrogen signaling in breast cancer.

Chan CS, Song JS CCCTC-binding factor confines the distal action of estrogen receptor. Cancer Res — Zhao H, Dean A An insulator blocks spreading of histone acetylation and interferes with RNA polymerase II transfer between an enhancer and gene. Jackson DA, McDowell JC, Dean A Beta-globin locus control region HS2 and HS3 interact structurally and functionally.

Parnell TJ, Geyer PK Differences in insulator properties revealed by enhancer blocking assays on episomes. Mukhopadhyay R, Yu W, Whitehead J, Xu J, Lezcano M, et al. TRβ-deficiency KO impairs T3-stimulated changes in most induced and repressed genes.

Unimpaired genes in the KO are listed at the bottom 56 and genes. b Dot plots of representative T3-responsive genes determined by RNA-seq and qPCR qPCR groups, 4 pools of 3 pituitaries.

The limited impairment in Thrb -KO mice of some T3-responsive genes, such as Tshb presumably reflects compensation by the T3 receptor TRα1 encoded by the Thra gene 13 , To support a role for T3-regulated pituitary chromatin sites as enhancers, we investigated the association of T3-dependent gene expression with T3-regulated chromatin sites.

In marked contrast, T3-depleted ATAC peaks were rarely associated with T3-induced or T3-suppressed genes.

Thus, T3-induced genes are associated with TRβ binding and T3-increased open chromatin characteristics of putative positive enhancers. However, T3-suppressed genes display a weaker association with TRβ-binding and poor association with chromatin opening or closing, precluding simple generalizations about characteristics of negative enhancers for T3-mediated repression.

Analysis of maintained TRβ-HAB sites; T3-increased ATAC sites and T3-depleted ATAC sites Fig. c Example of the T3-induced Gh gene showing TRβ-bound sites red arrowheads , T3-regulated open chromatin ATAC and histone marks.

In contrast, the strongly T3-suppressed Cga gene lacks obvious regulation of chromatin by T3 or detectable TRβ binding. Raw reads scale on left , below each mark label.

As an example, the growth hormone gene Gh , a T3-inducible gene, displays TRβ-bound sites with T3-inducible chromatin opening and extensive histone modifications Fig. The TRβ-bound site nearest the gene is consistent with in vitro transactivation studies of the rat Gh promoter 1 , In contrast, some T3-suppressed genes including the gene for the alpha subunit of TSH Cga lacked detectable TRβ binding or T3-regulated open chromatin despite the powerful suppression of Cga expression by T3 Fig.

We demonstrated T3-inducible enhancer activity for representative TRβ-bound sites in transactivation assays using luciferase reporters in transfected cells. Supplementary Fig. We further tested the role of DR4 motifs predicted by the genome-wide motif analysis. Mutagenesis of DR4 motifs in the Ceacam16 and Thrb sites showed that these motifs were essential for T3-inducible activity.

Our findings identify T3-inducible chromatin opening at a specific subset of TRβ-bound chromatin sites suggesting a key property of certain putative T3-sensitive enhancers in the pituitary gland.

We tested the hypothesis that T3-regulated gene expression is associated with inducible chromatin opening at putative enhancers by comparing equal groups of T3-inducible versus constitutively open sites Fig. However, genome-wide analysis showed a stronger association of T3-activated genes with inducible rather than constitutively open ATAC peaks Fig.

a Comparison of T3-inducible versus constitutively open chromatin ATAC peaks at T3-sensitive, putative enhancer sites in the pituitary gland. Analysis of 1, inducible ATAC sites versus the top constitutive ATAC peaks of non-regulated ATAC peaks.

Both groups maintain TRβ binding regardless of T3 status and have DR4 as the top motif. b Box plot of distribution of T3-regulated genes RNA level for both groups of ATAC sites. Statistical analysis by Mann—Whitney test. c Simplified model for a T3-activated pituitary enhancer. Receptor-bound enhancers, typically at distal sites, dynamically and reversibly control chromatin opening and histone modifications according to the T3 level.

We find that TRβ binding sites in pituitary chromatin are determined at both tissue- and gene-specific levels. Tissue-specific determinants may include cell type-specific transcription factors that stimulate chromatin opening or otherwise facilitate T3 receptor binding 2.

In the pituitary, the Gh gene is highly induced by powerful pituitary cell lineage activators such as Pit1, which may initiate activation and chromatin opening 1 , Although the T3 receptor may be a later-stage regulator, it mediates critical adjustable control over levels of Gh expression in response to T3.

We find that T3 induces extensive chromatin remodeling and histone acetylation around the Gh locus potentially involving an interplay of T3-regulated enhancers and complex upstream control regions The findings suggest that impaired chromatin regulation contributes to GH-deficiency in hypothyroidism 3 , 5.

A second level of specificity resides within the pituitary genome itself as TRβ binding sites are not equally responsive to T3. T3 induces chromatin opening and histone modifications primarily at sites that maintain TRβ binding regardless of T3 status. These sites represent the predominant category of receptor-occupied site in untreated conditions with physiological T3 concentrations.

In contrast, sites at which binding is determined by T3, that is de novo induced or abolished, show comparatively little modification of chromatin by T3. We speculate that persistent receptor-enhancer interactions, perhaps aided by a high incidence of near-optimal DR4 sequence motifs, create a poised state capable of dynamic responses to T3 fluctuations Fig.

The moderate, relative increase in TRβ binding at some of these sites might reflect further stabilization by T3-induced conformational changes as the enhancer shifts to a more active state.

The in vivo tissue data available suggest that shifts in receptor binding are a general response to T3 although the patterns vary. In pituitary tissue, the maintained sites represent the majority of occupied sites when T3 is in physiological ranges.

Potentially differing from our findings, a study of the liver reported that most receptor binding reflects T3-induced de novo recruitment based on chromatin immunoprecipitation with an antibody against T3 receptors encoded by both Thrb and Thra genes i.

However, another liver study using a knockin tag on TRβ1 reported that ligand increases the degree of binding rather than de novo binding at many sites An immunoprecipitation study of a human thyroid cell line in culture suggests that most receptor binding reflects T3-stimulated occupancy However, differences in experimental design, bioinformatics criteria, and technical detection of low levels of receptor binding preclude generalized conclusions between studies at the present time.

Most T3-dependent pituitary genes display some degree of resistance to T3 in Thrb KO mice indicating that TRβ mediates transcriptional sensitivity. The full phenotype is probably partly masked by TRα1 substituting for TRβ 13 , Current views of pituitary responses to T3 often focus on GH and TSH as clinically relevant, endpoint hormones in the circulation 5 , 9 but our identification of several hundred T3-responsive genes suggests a broader spectrum of primary transcriptional actions at the genomic level.

For T3-induced genes, our results support a model of direct activation and chromatin opening at TRβ-bound sites see later discussion. However, for T3-suppressed genes, low associations with T3-regulated open chromatin or TRβ binding suggest less obvious means of control.

Mechanisms of repression by T3 present a long-standing puzzle. Hypothetical negative enhancers might be expected to display chromatin closing at receptor-bound sites commensurate with the extent of gene suppression by T3.

However, some potently suppressed pituitary genes such as Tshb , Cga TSH subunits , and Trhr TRH receptor Fig. These findings may support long distance or indirect modes of suppression 47 , 48 and challenge earlier proposals of direct repression of Tshb and Cga based on transfection assays of short promoter fragments in vitro 49 , Although previous studies of short promoter fragments implied that negative enhancers reside close to the transcription start site, at the genome-wide level, our analysis did not reveal obvious enrichment of T3-regulated chromatin sites in the promoter-proximal region of T3-suppressed genes Fig.

Our in vivo evidence raises the possibility that T3-mediated pituitary gene suppression involves specialized forms of genomic control not involving obvious changes in chromatin accessibility or commonly studied histone modifications. Future studies might investigate other types of histone modifications, including active or repressive marks.

There may be varied means of negative regulation. A rare example of a negatively regulated gene that displays some, modest chromatin closing at a receptor-bound site is Opn1sw S opsin in the retina A liver study using tagged TRβ1 suggested that binding sites associate with both induced and repressed genes but with shifting cofactor interactions in each case A functional role for any site in mediating gene repression by T3 in vivo remains to be demonstrated.

We propose a model for pituitary gene activation in which poised receptor-enhancer complexes adapt dynamically to T3 levels Fig.

This model could explain the sensitivity of the pituitary to T3 since persistently bound, poised receptors could confer immediacy of response to T3 fluctuations. Our findings are compatible with views that T3 stimulates histone acetylation 25 , 26 , 28 , 29 , 51 but also implicate opening of the nucleosome array at poised sites in pituitary chromatin.

It is unclear if TRβ can bind to a pituitary enhancer in a fully closed chromatin state. Limited, pre-existing opening, perhaps primed by cell-specific factors Fig. Rising T3 levels may stimulate TRβ-bound sites to open further by recruitment of remodeling factors that displace nucleosomes 33 , allowing association with mediator and activation complexes A key feature of a poised receptor model is its reversibility by declining T3 levels which would close chromatin, deacetylate histones and reduce gene expression in hypothyroidism.

This proposal focuses on receptor-bound enhancers with T3-inducible open chromatin but does not exclude a role for other categories of binding sites. Future studies might test the hypothesis that in the pituitary, T3 shifts the association of receptor-enhancer complexes with histone modifying or remodeling factors.

However, the in vivo picture is far from complete because of limited in vivo tissue data and a lack of comparable analyses between available studies. We anticipate that there is not a single, uniform type of T3-regulated enhancer and that tissue-specific specializations may reflect functional adaptations for each tissue.

Knockin tags are increasingly used as a powerful tool to isolate transcription factors and bypass a lack of specific reagents for chromatin-binding studies. Any tag may disturb the function of the gene, or the activity or stability of the product, even if only subtly, which is difficult to exclude entirely.

For this reason, we employed an independent screen for enhancers by analysis of T3-regulated open chromatin, providing mutual support for findings obtained using the TRβ-HAB model. Mutations within the TRβ C-terminus as occur in human resistance to thyroid hormone disrupt T3-dependent transactivation 54 , However, this is not the case with the HAB tag which extends beyond but does not change the C-terminus or the AF2 activation domain of TRβ.

Interestingly, variable amino acid residues that extend beyond the AF2 domain are not conserved between nuclear receptors 56 , 57 and form a disordered extension in a crystal structure of TRα1 57 , which may explain why the HAB tag does not inhibit transactivation.

The similar T3-sensitive transactivation by tagged or non-tagged receptors Supplementary Fig. The utility of the TRβ-HAB model is supported by the consistency of chromatin binding sites identified in the liver using an N-terminal HA-TRβ1 tag 29 or the C-terminal TRβ-HAB tag Supplementary Fig.

Thus, tag location and different pull-down methods immunoprecipitation or affinity-purification do not overtly distort outcomes. Further support is provided by the finding of concordant sites in lipogenic genes in the liver using either virally expressed TRβ1 with an N-terminal biotinylation tag or the TRβ-HAB model Overall, our findings of dynamic control of pituitary chromatin by T3 suggest a genomic basis for understanding pituitary function and dysfunction as well as responses of the pituitary to widely used treatments for hyperthyroidism or hypothyroidism 9.

The Thrb HAB allele expresses TRβ proteins TRβ1 and TRβ2 fused to a peptide with a hemagglutinin HAx2 tag and a site for biotinylation by prokaryotic BirA ligase, modified from a published tag The tag was inserted at the endogenous Thrb gene by homologous recombination in W9.

The construct included a self-excising ACN neomycin-resistance cassette 59 , a 3. Targeting was confirmed by Southern blot and sequencing analyses. Genotyping primers and other primers used in this study are listed in Supplementary Table 3.

Genotyping was performed using PCR protocols provided by the Jackson Lab. Unless otherwise noted, adult male groups 2—4 months old were analyzed. To induce hypothyroidism, 0. Subgroups were made hyperthyroid by co-administration of 0. Studies were performed in accordance with the NIH Guide for Care and Use of Laboratory Animals and protocols approved by NIDDK Animal Care and Use Committee.

Antibodies are listed in Supplementary Table 4. Images were captured on a Leica SPE2 confocal microscope and processed using ImageJ. Pituitary glands were sectioned in the coronal plane and areas of a half-lobe of the anterior pituitary after immunostaining for GH were measured using ImageJ.

Areas of colloid within follicles 20 views and of total lobes 10 views were measured in transverse, mid-lobe sections using ImageJ. Auditory-evoked brainstem responses were tested using established procedures on adult mice under avertin anesthesia TSH, total T4 and total T3 levels in serum samples were measured using Milliplex magnetic bead panels.

TSH and GH were measured using a Milliplex MAP Mouse Pituitary Magnetic Bead Panel Mouse MPTMAGK; MilliporeSigma and Millipore Luminex plate reader with Millipore Analyst Software MilliporeSigma Charcoal-stripped FBS was used with T3 added at stated final concentrations the day after transfection.

Genomic DNA fragments containing TRβ-binding sites were cloned into chromatin-forming pREP4-Luc2 reporter vector. Samples were analyzed in triplicate and experiments repeated at least twice.

Rabbit antiserum against TRβ1 or TRβ2 65 was used to detect tagged and untagged receptors. The secondary antibody was horseradish peroxidase-goat anti-rabbit IgG Cell Signaling Technology, Original scans of western blots with markers are included in Supplementary Fig.

Nuclear protein extracts were prepared 65 from pituitaries pools of 3 of the indicated genotypes Supplementary Fig. Rabbit antiserum against TRβ2 65 was used to detect tagged and untagged receptors. Secondary antibody was horseradish peroxidase-goat anti-rabbit IgG ThermoScientific G , Signal was detected using Pierce ECL Plus Western Blotting Substrate Thermo Scientific, Cat and Kodak Biomax film exposure.

Random hexamer primers were used to synthesize cDNA from total RNA using SuperScript III reverse transcriptase Life Technologies. Relative RNA levels were normalized to Actb as a reference gene.

See Supplementary Table 3 for primer sequences. A Mann—Whitney test was used in Fig. Tests and groups are noted in the figure legends. Statistical tests were performed using GraphPad Prism version 9.

Total RNA was prepared from pools of 3 frozen pituitaries per library, using TRIzol Reagent Invitrogen, Cat , then mRNA enriched using Dynabeads mRNA purification Kit Ambion Cat and quantified using Qubit RNA Broad-range assay Kit Thermo Fischer Scientific. Libraries were synthesized using SuperScript Double-Stranded cDNA Synthesis Kit Invitrogen and sequencing libraries generated using Illumina Truseq Cat or ThruPLEX DNA-seq Takara, R kits.

Samples were multiplex-sequenced on an Illumina HiSeq instrument at the NIDDK Genomics facility. Single-end 50 base reads were mapped to RefSeq mouse transcript database mm9 Build 37 using BBMap version The nuclear pellet was resuspended in 1. Beads were washed in dilution buffer To release DNA, proteinase K was added at final concentration 0.

DNA was collected using a magnet rack, purified by QIAquick PCR Purification Kit QIAGEN, and quantified using Qubit dsDNA HS Assay Kit Thermo Fisher Scientific, Q ChIP-seq libraries were generated using an Illumina Truseq kit Cat The ChAP-seq method was modified from published protocols All subsequent steps were as in the ChIP-seq protocol.

ChAP-seq libraries were generated using Illumina Truseq kit Cat Tissue was homogenized in 0. All steps were performed on ice with incubations using an end-to-end rotator.

Fragmented genomic DNA was recovered using MinElute spin columns Qiagen and amplified by five cycles of qPCR. Typically, 4—7 PCR cycles were added to the initial 5 cycles.

Amplified DNA was purified on AMPure XP beads Beckman A , analyzed on an Agilent Bioanalyzer and sequenced 50 base, single end on an Illumina HiSeq instrument. Sequencing reads were aligned to mm9 genome build using Biotie2.

Differential analysis was performed using edgeR 3. Transcription factor binding motifs were obtained from the HOMER database Gene annotation was based on the NCBI reference sequence database RefSeq for mouse genome assembly mm9. Gene expression heatmaps and dot plots generated from RNA-seq or qPCR data were created using R version 4.

IGV gene maps were created using Integrative Gene Viewer version 2. Gene ontology for TRβ-HAB or ATAC peaks was analyzed using GREAT version 4. We assessed the consistency of chromatin binding peaks detected with TRβ-HAB C-terminal tag, streptavidin-based ChAP and another knockin, HAx3-TRβ1 N-terminal tag, antibody-based ChIP 29 by analyses of datasets for liver 58 GEO access GSE and GSE, respectively.

See Supplementary Fig. A summary of genomic datasets generated in this study is listed in Supplementary Table 5. Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

All data that support the findings are available within the manuscript and the Supplementary Information. Numerical source data for the graphs in the manuscript are available in a Supplementary Data file. Genomic datasets generated in this work are available at GEO with accession GSE Zhu, X.

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The Selenium testNG gland regulates Thyroid Function Enhancers and Enhncers via secretion of thyroid hormones Thyroir thyroid follicular cells TFCs. Loss of TFCs, Enhacers cellular Enhanvers, autoimmune destruction or surgical resection, underlies hypothyroidism. Recovery of Enhanfers hormone levels Blackberry and peach salsa recipe transplantation of mature Thyroie derived Enhancdrs B vitamin foods cells in vitro holds great therapeutic promise. However, the utilization of in vitro derived tissue for regenerative medicine is restricted by the efficiency of differentiation protocols to generate mature organoids. Here, to improve the differentiation efficiency for thyroid organoids, we utilized single-cell RNA-Seq to chart the molecular steps undertaken by individual cells during the in vitro transformation of mouse embryonic stem cells to TFCs. Our single-cell atlas of mouse organoid systematically and comprehensively identifies, for the first time, the cell types generated during production of thyroid organoids. Using pseudotime analysis, we identify TGF-beta as a negative regulator of thyroid maturation in vitro.