There is widespread interest in the three-dimensional chromatin conformation of the genome and its impact on gene expression. However, these studies frequently do not consider parent-of-origin differences, such as genomic imprinting, which result in monoallelic expression. In addition, genome-wide allele-specific chromatin conformation associations have not been extensively explored. There are few accessible bioinformatic workflows for investigating allelic conformation differences and these require pre-phased haplotypes which are not widely available.IGF2-H19 locus. Other imprinted loci (DLK1 and SNRPN) are more variable and there is no “canonical imprinted 3D structure,” but we could detect allele-specific differences in A/B compartmentalization. Genome-wide, when topologically associating domains (TADs) are unbiasedly ranked according to their allele-specific contact frequencies, a set of allele-specific TADs could be defined. These occur in genomic regions of high sequence variation. In addition to imprinted genes, allele-specific TADs are also enriched for allele-specific expressed genes. We find loci that have not previously been identified as allele-specific expressed genes such as the bitter taste receptors (TAS2Rs).We developed a bioinformatic pipeline, “HiCFlow,” that performs haplotype assembly and visualization of parental chromatin architecture. We benchmarked the pipeline using prototype haplotype phased Hi-C data from GM12878 cells at three disease-associated imprinted gene clusters. Using Region Capture Hi-C and Hi-C data from human cell lines , we can robustly identify the known stable allele-specific interactions at the This study highlights the widespread differences in chromatin conformation between heterozygous loci and provides a new framework for understanding allele-specific expressed genes.The online version contains supplementary material available at 10.1186/s13059-023-02876-2. Higher-order chromatin conformation forms a scaffold upon which epigenetic mechanisms converge to regulate gene expression , 2. Manycis, they are referred to as imprinting control regions (ICRs) . The moIGF2-KCNQ1 locus, implicated in BWS and SRS, divides into two imprinted gene clusters, each regulated by a separate imprinting control region (ICR). The first is IGF2-H19, with its ICR (H19-DMR) methylated on the paternal allele. The second cluster has a maternally methylated ICR (KvDMR) at the promoter of the long non-coding RNA (lncRNA) KCNQ1OT1 gene [KCNQ1 and adjacent genes [SNRPN locus is implicated in Prader-Willi and Angelman syndromes. Imprinting at this region is regulated by a ~35-kb bipartite imprinting control region (PWS-IC and AS-IC). The PWS-IC section is methylated on the maternal allele and is the promoter for the pre-mRNA transcript for SNRPN, SNURF, and SNHG14, which is also a host transcript for several other long and short non-coding RNAs [DLK1-DIO3 locus is implicated in Temple and Kagami-Ogata syndromes and includes DLK1 and RTL1  and several maternally expressed ncRNAs , snoRNAs, and miRNAs [. Two DMRs, IG-DMR (located in the intergenic region between DLK1 and MEG3) and MEG3-DMR (at the MEG3 promoter), regulate imprinted expression at this locus [The OT1 gene  that whent genes . The SNRing RNAs . The DLKd miRNAs . Two DMRis locus . These DIGF2-H19 locus regulates access to a series of enhancers through allele-specific differences in higher-order looping interactions [Imprinted genes are an excellent model system for analyzing epigenetic regulation of gene expression and the study of genomic imprinting has uncovered many paradigms that are generally relevant to gene expression . One sucractions –32.These early studies used a chromosome conformation capture (3C) technique in which fixed chromatin is digested with a restriction enzyme followed by a ligation reaction that favors regions in close proximity. The principle of 3C technology is that interactions between distant regulatory regions that come close together in the 3D space will be more frequently detected than random interactions . Newer tTADs are thought to regulate gene expression by increasing the frequency of intra-domain promoter-enhancer interactions and insulating against spurious inter-domain interactions. TADs are formed via cohesin-mediated loop extrusion, whereby DNA is bidirectionally extruded through the ring-shaped cohesin complex until it is halted by convergently oriented CTCF to form a TAD boundary .It is further assumed that CTCF and associated protein TAD boundaries compartmentalize the genome to implicitly prevent transcription read-through and spurious transcriptional activation of silent genes or constrain the spread of silencing chromatin –42. ParaIgf2-H19, Dlk1-Dio3 imprinted loci. For these loci in the mouse, it has been shown that the maternally and paternally imprinted genes are located together in large TADs that are similar in both parental alleles. Within the TADs, differential binding of CTCF creates allele-specific subTAD structures that provide the instructive or permissive context for imprinted gene activation during development [Mouse models in which an imprinted locus can be deleted and transmitted through either the male or female germline, have enabled allele-specific Hi-C profiles for the elopment .cis interactions fortuitously link single-nucleotide polymorphism (SNP) variants within a chromosome and provide molecular haplotype information. One of the first studies to use haplotype phasing in Hi-C data from a human lymphoblastoid cell line, GM12878, detected allele-specific long-range interactions between a distal locus, termed HIDAD  and the promoters of the maternally expressed H19 and the paternally expressed IGF2 [IGF2-H19 has been studied in great depth as the archetypal locus for allele-specific interactions [IGF2-H19 is not universally true for all imprinted gene clusters.A limitation to studying imprinted genes in humans has been the need for family studies to ascertain the parental origin of genes. Technologies that detect long-range sed IGF2 . IGF2-H1ractions , 45. HowIn this study, we sought to examine how the higher-order chromatin conformation structures differ between the active and silent alleles at loci containing genes that are allele-specifically expressed in humans. To this end, we assembled a HiCFlow pipeline for processing raw Hi-C data for haplotype phasing and construction of allele-specific chromatin conformation profiles. A number of existing pipelines, including HiC-Pro , are capIGF2-H19 locus, the ICR plays a direct role in directing allele-specific CTCF-mediated higher-order chromatin structures consistent with loop extrusion models, whereas at other loci, the ICR may have indirect or no specific effect. Allele-specific compartmentalization was observed in some cell lines at the SNRPN and DLK1 loci. Rather than remaining spatially and temporally separated from their non-imprinted neighbouring genes, imprinted gene clusters share TADs with non-imprinted genes. Indeed, most allele-specific interactions occur within subTADs. Imprinted domain boundaries may be delimited by TAD structures, but some allele-specific associations can occur across TAD boundaries with concomitant effects of allele-specific expression. Allele-specific interactions were not confined to imprinted domains. In an unbiased genome-wide screen, we detected additional allele-specific TADs (ASTADs). The ASTAD distribution varied between cell lines. We found 8–32% of genes with allele-specific expression to be located within ASTADs. Regions of high genetic variability, such as olfactory receptor loci, the bitter taste receptor (TAS2R) gene cluster and the keratin gene (KRT) cluster, were found to be within ASTADs.Our analyses indicate that imprinted gene domains are not uniformly organized within a canonical higher-order structural profile regulated by elements within the ICRs. At the MboI), we generated capture regions  at 5 imprinted chromosomal loci, the largest regions included SNRPN, DLK1-DIO3, and IGF2-KCNQ1  dataset in a human breast epithelial cell line, 1-7HB2. This diploid cell line has previously been used to examine allele-specific expression and imprinted methylation for several imprinted genes , 47–49 aIGF2-H19 locus to benchmark the allele-specific subtraction parameters and confirmed that the known allele associations could be robustly detected , SNRPN (Prader-Willi Angelman (PWS-AS) locus, chromosome 15q11-q13), and DLK1-DIO3 (chromosome 14q32.2) loci  forms a weak subTAD boundary. The difference in allele-specific enhancer interactions with IGF2  are towards the left, whereas A1 (blue) are towards the right. We have highlighted these as triangles with dotted outlines  are within this TAD and appear to form a subTAD boundary  that regulates CTCF site availability, this is not invariably the case at other loci. Indeed, at the SNRPN locus, TAD-like structures are assembled in the absence of CTCF sites, and independent of the ICR.It has been proposed that ICRs such as the above DMRs, mediate their epigenetic functions by directing allele-specific chromatin conformation. However, not all imprinted genes contain CTCF sites at their ICRs . Allele-To further examine whether known ICRs are anchor points for allele-specific chromatin interactions, we conducted allele-specific viewpoint analyses using haplotype phased Hi-C data from the following cell lines GM12878, IMR-90, and H1-hESC, alongside our 1-7HB2 library. We compared these to the relevant subtraction matrices, to which we also added Peakachu loops . ViewpoiIGF2-H19 and the KCNQ1 loci. In GM12878 cells, the maternal origin of the H19 interactions with the downstream HIDAD locus and the reciprocal paternal IGF2-HIDAD interactions have previously been demonstrated [H19-DMR and maternal interactions at the IGF2 promoter regions as A2 (red) in the subtraction matrices for all three cell lines. This enabled us to also assign the parental origin to the nearby KCNQ1 locus. Figure H19 and KCNQ1 loci. We note that the H19-DMR strongly anchors the maternal allele-specific interactions as can be seen in the subtraction matrices , while in H1-hESCs the ICR interacts with regions further downstream  has a weaker and more variable effect on the higher-order structure  approximately −100kb of the KvDMR  and CDKN1C   is present in all these cell lines, but at a lower level in IMR-90. The bipartite imprinting center at the SNRPN locus therefore seems to affect allele-specific chromatin conformation at the wider locus. This effect is not reliant on the methylation status of the DMR region within the ICR, at least not in H1-hESC cells. The substantial allelic interaction differences in H1-hESC, despite hypermethylation of the DMR, may reflect the stability of imprinted gene expression of this locus in embryonic stem cells which occurs independently of DNA methylation [At the hylation , 62.DLK1-DIO3 locus, only IMR-90 and 1-7HB2 cells showed allele-specific associations with the IG-DMR viewpoints . There may also be a stripe in the opposite direction; however, this is less consistent between cell lines. The MEG3-DMR contains CTCF sites, which have been reported to be important in maintaining imprinting in somatic tissues [DLK1, MEG3, MEG8 RTL1, or DIO3 in these cell lines. Indeed, 1-7HB2 and IMR-90 which had the lowest level of expression for these genes showed the strongest allele-specific loops, and more intense differences between A1 and A2 on the subtraction matrix  data to provide the mean methylation level per CpG within the region   to our HIGF2-H19 locus, allele-specific interactions did not extend beyond the HIDAD region  in our cell lines , that were not associated with any genes and a 304-bp duplication in an intron of the VRK1 gene , listed in the NCBI database. In our cell lines, we find no variation in copy number within the v-TAD domain. However, both GM12878 and H1-hESC possess different Indel mutations within the region corresponding to the nssv16165643 duplication. It is unclear to what extent these variants are responsible for allele-specific associations in the v-TAD. Since they do not overlap CTCF sites, we do not anticipate they are responsible for such large-scale allelic changes in GM12878 and H1-hESC.All cell lines have a large 3-Mb-sized TAD corresponding to a B-compartment that is located 2Mb upstream of the VRK1 encodes a Serine/Threonine Kinase and is associated with pontocerebellar hypoplasia, Type 1A and Microcephaly-Complex Motor and Sensory Axonal Neuropathy Syndromes. It is widely expressed in several tissues and has roles in cell cycle, mitosis and DNA damage responses. It has never been reported to have monoallelic expression. We found it to be highly expressed in all cell lines  were found to have allele-specific expression , 5.5–9.1 (DLK1-DIO3 in IMR-90), and the SNRPN locus (2.2-3.1 in H1-hESCs). The v-TAD described above had a Z-score >4 in GM12878 and H1-hESC.The detection of the above v-TAD prompted us to examine the frequency in which differences in allele-specific associations can be found within TADs genome-wide. We therefore performed an unbiased ranking of all TADs to assess the allelic association differences genome-wide in GM12878, IMR-90, and H1-hESC cells Fig. a. We defEach cell line had its own unique profile of ASTADs distributed across the genome  or genomic  features, we carried out locus overlap analysis (LOLA)  for 15 sCNVs are known to influence chromatin architecture  and therThe three cell lines tested came from three different individuals and represent three different cellular lineages . Based on these genetic and the tissue differences, we expect only a few ASTADs would be common to all three cell lines. From the above analysis, we found 39 conserved ASTAD. We hypothesized that common ASTADs would fall into two categories. The first category being sequence directed, such that ASTADs present at the same location in the three cell lines would share identical SNPs variants. The second category being ASTADs with stable epigenetic mechanisms directing allele-specific chromatin conformation as in genomic imprinting.n = 10,000) of conserved TAD versus ASTAD boundaries  of identical genetic variants at conserved ASTAD boundaries compared to conserved TADs.We first tested whether conserved ASTAD boundaries possessed the same, i.e., identical genetic variants, across all three cell lines that may influence chromatin structure in a consistent manner. Randomization testing with repeat random sampling n = 0,000 of Z-score = 1,67, p = 0.048). These results suggest that conserved ASTADs are primarily sequence directed due to sharing similar haplotypes.A similar analysis, examining the distribution of allele-specific methylation (ASM) within conserved TAD versus ASTAD boundaries was performed for each cell line. We did not detect any significant enrichment of in H1-hESC and IMR-90. Enrichment was marginally higher in GM12878 (Z-score = 4.9–9.7), was identified at chr3:195,270,000–195,730,000  are not within blacklisted regions.The conserved ASTAD with the highest ranking allelic difference  and a feature of such loci are that the SNPs have longer linkage disequilibrium (LD) ranges than random SNPs . KRT1 haorphisms , and a morphisms . In GM12H19, IGF2, IGF2-AS and INS) are part of the same imprinted gene cluster on chromosome 11, the other is the recently identified pseudo-gene ATP5F1EP2 [IGF2-H19 cluster is the most studied of imprinted genes, and perhaps this is due to its robust and stable CTCF-mediated imprinting mechanism. Little is known about imprinting mechanisms for ATP5F1EP2. However, a close look at this locus showed allele-specific expression of further genes within this ASTAD, including POLR1D, MTIF3, and USP12 in H1-hESC, and RPL21 in GM12878. These genes have not been reported to be imprinted. Gene mutations in POLR1D underlie autosomal dominant inheritance of Treacher Collins Syndrome .Only 5 Imprinted genes are present in conserved ASTADs. Four of these  within ASTADs compared to non-ASTADs  within ASTADs in GM12878 and IMR-90, but not in H1-hESC , IMR-90  and H1-hESC .ASTADs are domains with high frequency of allele-specific contacts. Therefore, we examined whether genes located within ASTADs have allele-specific expression and downloaded RNAseq ASE data for GM12878   and IMR-p < 0.001) in GM12878 and IMR-90, but not in H1-hESC. In addition, we find that enhancers associated with ASE genes are significantly over-represented in ASTADs  in GM12878  are enrTRIM genes on chromosome 11 contained allele-specific  expressed genes  and were within an ASTAD in GM12878, and in IMR-90 , which typically express only one allele, but did not feature in the lists of ASE in these cell lines. Olfactory genes have been shown to form interchromosomal associations and aggregate in foci within the nucleus when they are repressed, with the expressed allele localized outside of such foci [We further found that a cluster of uch foci , 81.TAS2R gene cluster that encodes an array of Bitter Taste Receptor genes on chromosome 12p13.2. The TAS2R gene cluster overlaps an ASTAD in both GM12878  and in IMR-90  corresponding to an overlap of approximately 90%, but this may be due to a lack of SNP density in IMR-90  and 7 (TAS2R3-TAS2R38) and found that these did not overlap ASTADs. However, to our knowledge this is the first time that taste receptors on chromosome 12 have been reported to be present in an allele-specific chromatin conformation.One ASTAD region of interest included the The recent attempts to link chromatin interaction information with GWAS variants to target genes has led to several tools being developed to predict the functional effects of variants in disease . HoweverIn this study, we assembled a novel bioinformatics pipeline, HiCFlow, which combines variant calling and haplotype phasing with allele-specific Hi-C analysis to enable robust investigation and visualization of allele-specific associations. Since genomic imprinting is a phenomenon of epigenetically established parent-of-origin monoallelic gene expression, that is independent of the genetic sequence of the expressed or silenced allele, we initially focused on these genes as domains known to be allele-specifically regulated.IGF2-H19 is consistent with an allele-specific loop extrusion model, which shows up as a pair of parallel stripes in a subtraction matrix. In this case, CTCF sites anchored at the HIDAD locus (or other sites downstream in a tissue-specific manner) interact with CTCF sites at the H19-DMR on the maternal allele, and with CTCF sites near the IGF2 promoter on the paternal allele.We focused on three imprinted gene clusters that exemplified imprinted domains known to be allele-specifically regulated. The canonical boundary model exemplified by IGF2-H19 is consistent with an allele-specific loop extrusion chromatin conformation model, this is not invariably the rule at all imprinted loci. Allele-specific loop extrusion shows up as a pair of parallel stripes in a subtraction matrix at the IGF2-H19 locus and it is the H19-ICR that determines the allele-specific chromatin scaffold. Here, CTCF sites at the HIDAD region and 9 other intervening CTCF pause-sites engage in loop extrusion, either with the CTCF-rich H19-DMR on the maternal allele, or with CTCF sites near the IGF2 promoter on the paternal allele. A large body of literature exists for the IGF2-H19 locus, demonstrating that deletion of the ICR, or its inactivation through DNA methylation, results in loss of imprinting, with over expression of IGF2 causing BWS [KCNQ1 locus has weak if any effects on 3D chromatin structure. If there is allele-specific loop extrusion, this occurs at a CTCF site previously identified as “region 3” and shown to be required for setting up a maternal allele-specific loop to facilitate KCNQ1 expression [KCNQ1OT1 lncRNA transcript is regulated by the KvDMR, and its transcription may disrupt CTCF binding at region 3 on the paternal allele to prevent this loop. RNA polymerase 2 has previously been reported to displace CTCF occupancy [KCNQ1 locus, and even if it did, the ICR would still have an indirect effect on 3D structure at this locus.The key finding at imprinted loci is that although the canonical boundary model for regulating imprinted genes expression as exemplified by sing BWS , 82–86. sing BWS , 82–85. pression . The patccupancy . HoweverSNRPN locus demonstrated that active and silent alleles occupy different nuclear compartments [IGF2-H19 and the KCNQ1 loci in the cell lines tested. Strong differences in compartmentalization were observed at the DLK1 locus in IMR-90 cells. This correlated with ICR methylation levels that would be consistent with normal imprinting and strong allele-specific association differences. A striking allelic difference in compartment structure was detected at the SNRPN locus in H1-hESC which also showed clear evidence of CTCF occupancy at the locus. Current models suggest that CTCF stabilizes TAD structures and that compartmentalization processes counteract the formation of TADs [SNRPN locus suggest that CTCF stabilized higher-order structures may also be initiated by phase condensation-mediated compartmentalization. Indeed, recent analysis suggests that CTCF may have an instructive function in the formation of condensates [Igf2 and Meg2 loci [Phase condensation models have not previously been tested at imprinted gene loci. Data from chromatin immunoprecipitation for post-translational histone modification profiles at imprinted loci in mouse and humans  predict artments . Adding eg2 loci . The locOur novel HiCFlow pipeline is suitable for processing multi-sample Hi-C, RC-Hi-C and Micro-C datasets. It is implemented as a user-friendly Snakemake pipeline and, to our knowledge, is the first workflow to combine haplotype phasing with allele-specific-Hi-C analysis. Testing it on a RC-Hi-C dataset provides proof-of-concept data for a diagnostic assay that can be used to detect allele-specific chromatin conformation in humans with imprinting disease. In this capture data set, we only examined a small set of imprinted gene clusters, but these could be expanded to the full set of imprinted genes and would still provide the required read depth when sequenced in-house on standard sequencing platforms. A limitation of our RC-Hi-C analysis was that we focused on the core regions of the imprinted domains, which enriched for the detection of shorter-range interactions. We would recommend utilizing tools, such as CHiCANE and Peaky, to detect long-range interactions , 92. ForTo investigate the properties of genome-wide allele-specific interactions, we scored each TAD based on the absolute allelic differences observed. The set of top-ranked TADs, denoted “allele-specific TADs (ASTADs),” were assessed for over-representation of various genomic annotations relative to non-ASTADs. Most strikingly, ASTADs were found to be enriched with polymorphic variants. This is unsurprising since heterozygous SNPs are necessary to distinguish alleles during allelic assignment of read pairs. However, enrichment of INDELs in ASTADs, which were not used for allelic assignment, suggests that high genetic variability plays a role in influencing allele-specific chromatin conformation. Indeed, regions of high variability are prone to allele-specific gene expression as demonstrated by the large body of GWAS studies that correlate genetic variants with allele-specific binding of transcription factors, DNA methylation patterns and gene expression (reviewed ). This iWe also found that allele-specific expressed genes were significantly over-represented in ASTADs in GM12878 (32%) and IMR-90 (18%), although not in H1-hESC (8%). Despite enrichment, only a small absolute proportion of ASE genes overlapped ASTADs. In some cases, it is likely that short-range allele-specific interactions may be indistinguishable at the 20kb resolution of our data. In addition, the absence of informative SNPs can prevent ASE detection and detection of allele-specific chromatin interactions.H19/IGF2 locus was consistently identified as an ASTAD in all cell lines. Allele-specific associations at imprinted loci also did not correlate with levels of detectable transcripts. It has been suggested that the chromatin conformation forms a scaffold upon which transcription factors can dock and activate gene expression [As expected, imprinted genes were significantly over-represented in ASTADs compared to non-ASTADs. However, only the pression . Such a pression . A lack pression , 97. ThuTAS2R family of receptors similar to olfactory receptors are G-protein coupled receptors. We were intrigued to find that the bitter taste receptor (TAS2R) clusters were present within ASTADs. To our knowledge, TAS2R genes have not been reported to be subject to allelic exclusion, unlike olfactory receptors. They occur in regions of high genetic variability similar to olfactory receptors. There are about 25 functional TAS2R genes and 11 pseudogenes spread between chromosomes 5, 7, and 11. Extensive population studies have utilized the high variation to examine evolutionary origins of different haplotypes and to identify the selection pressures that an ability to distinguish bitter toxic substances has had on the genetic evolution of this gene family. The functional effects of the variants on G-protein receptor protein structures and the mechanisms whereby they convey taste perception to the brain have been elucidated. However, limited information on their transcriptional regulation exists. It has been recently shown that TAS2R genes are not only expressed on the tongue but that they are more widespread and present in heart and respiratory epithelia as well as in the gut and that they may have further sensing functions unrelated to bitter taste. An in situ hybridization study has indicated that humans co-express a heterogeneous mix of between 4 and 11 taste receptors per cell in papillae of the tongue. Although we do not know if this is due to allele-specific expression, our data indicate that this may be the case as ASTADs are commonly associated with allele-specific expression.We were able to confirm that regions of high variability and known to have allelic imbalances in expression such as the olfactory receptor genes were within ASTADs. The This study exemplifies the utility of the bioinformatics HiCFlow tool for combined variant calling and haplotype phasing with allele-specific Hi-C analysis for investigation of allele-specific associations at regions subjected to epigenetic silencing such as genomic imprinting as well as sequence-mediated influences on expression. Overall, this study highlights how genetic sequence variation and the regulatory mechanisms behind allele-specific gene regulation culminate in widespread allelic differences in chromatin organization that are not confined to imprinted gene loci.Human mammary epithelial cell (1-7HB2) line were purchased from the ECACC  (Culture Collections). 1-7HB2 were cultured in RPMI-1640 (Sigma-Aldrich), supplemented with 5% fetal bovine serum , 10 ml/l penicillin-streptomycin solution , 5 μg/ml insulin , and 1 μg/ml hydrocortisone . GM12878 cells (B-Lymphocyte) were purchased from Coriell Institute and cultured in RPMI-1640 (Sigma-Aldrich) supplemented with 15% fetal bovine serum . IMR-90 cells  were obtained from ATCC (CCL-186™). Cell lines were cultured at 37°C. All cell lines have been periodically tested in-house for mycoplasma contamination.7 number of 1-7HB2 cells were crosslinked on plate with formaldehyde (Agar Scientific R1026), followed by a quenching step with 1.25M glycine, scraping for cell detachment and two washes with cold PBS 1×. The cell pellet was re-suspended in 50ml freshly prepared ice-cold lysis buffer , one protease inhibitor cocktail tablet ). Cells were lysed on ice for a total of 30min, with 2 × 10 strokes of a Dounce homogenizer with a 5-min break between Douncing to minimize cell clumping. Following lysis, the nuclei were pelleted and washed with cold 1.25xNEB Buffer 2  then re-suspended in 1.25xNEB Buffer 2 to make two aliquots of 10–15×106 cells for digestion. Hi-C libraries were digested using 1500U MBOI  at 37°C overnight while orbital shaking. Following digestion, the restriction fragment overhangs are filled in for 1h with dNTPs including biotin-14-dATP . Fragments are then blunt-end ligated with 1U/μl T4 DNA ligase  under dilute conditions to favor ligation between crosslinked fragments (in a 15-ml tube for overnight at 16°C). DNA crosslinks were then reversed with 10mg/ml proteinase K  for 6–8 h at 65°C followed by RNase A  treatment at 37°C for 60 min. Two rounds of DNA extraction/purification were carried out with phenol pH 8.0  and phenol: chloroform: isoamyl alcohol  followed by precipitation with 3M sodium acetate pH 5.2  and 2.5× volume of ice-cold 100% ethanol on wet ice for 1–2 h. The Hi-C Library’s quantity and quality were assessed by running 50–100ng of the Hi-C libraries on a 0.8% agarose gel. Hi-C marking and Hi-C ligation efficiency was verified by PCR digest assay using MBOI and CLAI  enzymes. Biotin is then removed from the ends of un-ligated fragments using the exonuclease properties of T4 DNA polymerase , and DNA was sheared to obtain DNA fragments with a peak concentration around 400 bp. DNA ends were repaired and fragments, with internally incorporated biotin, are pulled down using magnetic Dynabeads MyOne Streptavidin T1 beads (Life Technologies 65601). After PE adaptor ligation ((5′-P-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG-3′ and 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3), pre-Capture amplification was performed with eight cycles of PCR on multiple parallel reactions from Hi-C libraries immobilized on Streptavidin beads, which were pooled post PCR and SPRI Ampure XP beads  purified. The final Hi-C library was re-suspended in 25μl of Tris low-EDTA and quantified by the Qubit™ dsDNA BR Assay Kit . The size distribution of the library was assessed by Tapestation D1000 (Agilent). The Hi-C capture regions were enriched via hybridization with biotin-RNA probes (Agilent Technologies). The capture regions of interest were then pulled down with Dynabeads MyOne Streptavidin T1 beads (Life Technologies 65601) and purified using SPRI Ampure XP beads . Finally, Hi-C Capture library was amplified and then sequenced with Illumina 50 bp paired-end sequencing.3–4×10Capture biotinylated 120-mer RNA oligos  bases) were designed to target either one or both sides of MBOI site and within 4–500bp as close as possible to the ends of the targeted restriction fragments using a custom genome-wide Perl script made available from the Babraham Institute and then submitted to the Agilent eArray software (Agilent) for manufacture.RNA was extracted from cell lines using TRI Reagent (Sigma-Aldrich) and 1μg of total RNA was converted to cDNA using QuantiTect Reverse Transcription Kit (Qiagen). Quantitative PCR (qPCR) was performed using a ¼ dilution of cDNA with SYBR Green PCR Master Mix (Thermo Fisher Scientific) on ABI Step One Plus (Applied Biosystems) and specific primers (Sigma-Aldrich) for target genes  [Allele-specific gene expression data for IMR-90 and H1-hESC –108 were(GRCh37) . Genes wAllele-specific methylation datasets for GM12878 , IMR-90 IGF2-H19 locus in GRCh38. Since this locus is an essential control, we have herein presented all results using the GRCh37/hg19 reference. Alignment files were re-merged to paired-end files using Samtools (v1.1.0) [Hi-C data was processed using our in-house pipeline, HiCFlow. Read adapters were trimmed, using Cutadapt (v3.5)  and trun(v1.1.0) . Reads w(v1.1.0) . Finally(v1.1.0) .For allele-specific analysis, a phased haplotype for IMR-90 and H1-hESC was generated from the raw Hi-C data. Variants were called using the GATK (v4.2.4.1) best practises pipeline . In brieVisual comparison of allelic matrices A1 vs. A2) was performed using “subtraction matrices.” Joint-normalization of raw A1 and A2 matrices was first performed using HiCcompare (v1.6.0) . This pr was perfProcessing of RC-Hi-C was as described above, but analysis was restricted to read pairs mapping within a single capture region. All other read pairs were filtered from the analysis.Following sequencing, quality control of the raw FASTQ data sets was performed using FastQC (v0.11.9). Screening for potential sequence contamination was performed using FastQ Screen (v0.5.2) . ReproduHiCFlow performs compartment analysis using CscoreTool (v1.1) . CompartZ-score is calculated by comparing against the chromosome-wide background level of absolute differences for a domain of equivalent size. The methodology of ASTAD detection is illustrated in Fig. TAD domain detection was performed, using OnTAD (v1.2) , on the To determine if ASTADs were enriched for particular genomic features, we performed enrichment analysis using LOLA v1.22) . ASTAD e.22 . ASTOverlap analysis was performed to identify ASTADs that were conserved between cell lines. Conserved ASTADs were defined as sets of ASTAD intervals, between cell lines, with at least a 90% reciprocal overlap. A 10% difference in overlap allows for slight error in domain positioning due the loss of resolution during matrix binning. Given a bin size of 20kb, a 10% difference equates to a shift of approximately one bin length for a median size domain interval. Interval overlap was calculated using BedTools (v2.29.2).In each of the following randomization tests, any TAD domain overlapping a region with non-normal copy number or overlapping an Encode Blacklist region were removed from the enrichment analysis.n = 10,000) of conserved TAD boundaries. A TAD boundary was defined as the outermost 20kb of a TAD domain, equivalent to 1 bin size on the AS-Hi-C matrices. This analysis was repeated for the allele-specific methylation (ASM) data for each cell line  present in all three cell lines. The total number of observed conserved variants overlapping the set of conserved ASTAD boundaries was compared against a null distribution of repeat random samples n = 0,000 of Z-score from the observed overlap. This analysis was repeated for each cell line and for ASE genes  data, corresponding to CpG methylation in ENCODE bed bedMethyl format, were obtained from publicly available datasets . PCR amplification products of the bisulphite template using previously published primers for IG-DMR (hg38_chr14: 100810848-100811276) , KvDMR . Table S5. LOLA Enrichment “Max Rank” Score among 20 genomic features. Table S6. Oligo primers used in qPCR. Table S7. Proportion of ASTAD / non-ASTAD domains overlapping Normal CNV. Table S8. Observed vs. Expected Overlap of Features in Conserved ASTADs. Table S9. Observed vs. Expected Overlap of Genes in ASTADs.Additional file 2: Fig. S1. HiCFlow pipeline and Region Capture HiC (RC-HiC) library. Fig. S2. Comparison of subtraction matrices, at IGF2-KCNQ1 locus, between experimental validated and HiCFlow inferred haplotype in GM12878. Fig. S3. Supporting information relevant to Fig. Fig. S4. The effect of the PWS-AS imprinting control region on allele-specific chromatin conformation. Fig. S5. The effect of the IG-DMR/MEG3 imprinting control region on allele-specific chromatin conformation. Fig. S6. Compartment analysis of H19-KCNQ1 and SNRPN loci. Fig. S7. Compartment analysis of DLK1-DIO3 locus. Fig. S8. Features and distribution of ASTADs. Fig. S9. KRT gene cluster on chr12 is within a conserved ASTAD.Additional file 3: File S1. All Gene Statistical Analysis.Additional file 4: File S2. All TADs Statistical Analysis.Additional file 5: File S3. Conserved ASTADs Statistical Analysis.Additional file 6: File S4. Chi-square analysis.Additional file 7: File S5. Conserved SNPs.Additional file 8. Review history.


The molecular pathogenesis of pituitary neuroendocrine tumors (PitNETs) involves the interplay of many genes and transcription factors in the setting of a diverse altered tumor microenvironment. Recent advancements in molecular technologies, such as single-cell RNA sequencing, became essential in delineating specific cell types and identifying altered genes in both normal physiological states and diseases in the pituitary. This review discusses the recent progress made in characterizing the transcriptomic profiles of the normal pituitary gland and sporadic PitNETs and their significance.The pituitary gland is one of the most cellularly diverse regions of the brain. Recent advancements in transcriptomic biology, such as single-cell RNA sequencing, bring an unprecedented glimpse into the molecular composition of the pituitary, both in its normal physiological state and in disease. Deciphering the normal pituitary transcriptomic signatures provides a better insight into the ontological origin and development of five types of endocrine cells, a process involving complex cascades of transcription factors that are still being established. In parallel with these observations about normal pituitary development, recent transcriptomic findings on pituitary neuroendocrine tumors (PitNETs) demonstrate both preservations and changes in transcription factor expression patterns compared to those seen during gland development. Furthermore, recent studies also identify differentially expressed genes that drive various tumor behaviors, including hormone hypersecretion and tumor aggression. Understanding the comprehensive multiomic profiles of PitNETs is essential in developing molecular profile-based therapies for PitNETs not curable with current treatment modalities and could eventually help align PitNETs with the breakthroughs being made in applying precision medicine to other tumors.  The pituitary gland is composed of a heterogeneous population of cells that regulate essential homeostatic functions including metabolism, reproduction, and growth. The cell clusters in the anterior lobe of the pituitary gland are mainly characterized by the endocrine hormone that they produce and secrete into blood circulation. Corticotrophs produce adrenocorticotrophic hormone (ACTH), gonadotrophs produce follicular-stimulating hormone (FSH) and luteinizing hormone (LH), somatotrophs produce growth hormone (GH), and lactotrophs produce prolactin (PRL). All pituitary endocrine cells are tightly regulated by upstream hypothalamic neurons and assume different gene expression states in response to internal and external homeostatic cues ,2.Pituitary neuroendocrine tumors (PitNETs) (formerly pituitary adenomas) are common intracranial tumors originating from anterior pituitary neuroendocrine cells. PitNETs may cause intracranial mass effects, including headache, dizziness, visual deficits, or deficiency of pituitary hormones . Nearly Unlike hypersecreting PitNETs, nonfunctioning PitNETs may be found incidentally or present with signs of mass effect rather than symptoms of excessive hormone secretion. Some nonfunctioning PitNETs are “silent PitNETs” that are immunohistochemistry (IHC)-positive for anterior pituitary hormone and transcription factors, although they do not secrete hormones at a clinically significant level . While mMEN1 gene mutation in multiple endocrine neoplasia-1 (MEN1)) or as a familial isolated pituitary adenoma) [The genetic and molecular pathogenesis of PitNETs is diverse. Less than 5% of all PitNETs arise from germline mutations as a part of syndromic diseases  . The remadenoma) , with thDeciphering the molecular characteristics of PitNETs is of particular interest because such information could provide opportunities for unique personalized therapies. Unfortunately, understanding the comprehensive pathogenesis of PitNETs has been difficult mainly due to the interplay of many genes and unique, innate properties of anterior pituitary cell types in the setting of diverse altered tumor microenvironments . Recent TPIT , a T-box transcription factor, activates the hallmark gene POMC with the help of tissue-specific homeodomain transcription factor, PITX1 [TPIT in transgenic mice differentiates the pituitary corticotroph lineage, whereas TPIT double-knock down results in the loss of corticotroph cells [TPIT, the pituitary neuroendocrine cells that normally differentiate into corticotrophs differentiate instead into gonadotrophs or thyrotroph [TPIT suppresses altered cell lineage fates.During normal corticotroph development, r, PITX1 ,12  transcription factor gene, at the intermediate states of the corticotroph lineage [NEUROD1 may be required for the appropriate timing of corticotroph differentiation [NEUROD1 knock-down results in transient delayed corticotroph differentiation but no changes in TPIT expression [In the human fetal pituitary, scRNA-seq shows a peaked expression of  lineage . NEUROD1ntiation , althougntiation . In micepression . Thus, uMNX1  as the second highest significant transcription factor, following TPIT, in POMC cells [MNX1 and TPIT co-localizes via in situ mRNA hybridization in adult mice pituitary. MNX1 is involved in developing pancreatic beta cells [Furthermore, scRNA-seq of postmortem pituitaries of pediatric and adults identifies MC cells . In the ta cells  and motota cells , but itsPIT1 , a POU domain-containing transcription factor, is essential for the terminal cell differentiation and gene expression of normal pituitary cells that go on to secrete the neuroendocrine hormones PRL, GH, and TSH [PROP1 and negatively regulated by HESX1 [ and TSH . The forby HESX1 .NEUROD4 , a PIT1 target gene, is involved in cell maturation and proliferation. NEUROD4 double-knockout mice show specific downregulation of GH and growth hormone-releasing hormone receptor (GHRHR) [NEUROD4 knock-out mice may be transient. GH-positive cells, albeit in reduced numbers, are still detectable postnatally in the NEUROD4 knock-out mice, although the expression of GHRHR remains minimal [NEUROD4, similarly to NEUROD1 in corticotrophs, may regulate the appropriate timing of somatotroph differentiation rather than terminal cellular fate.In normal somatotrophs,  (GHRHR) . However minimal . These fFOXO1 and CEBPD in fetal human somatotrophs [FOXO1 in mice results in delayed terminal differentiation of somatotrophs and reduced expression of GHRHR [FOXO1 knock-out results in decreased expression of NEUROD4. Thus, FOXO1 may be an upstream regulator of NEUROD4 during somatotroph differentiation [NEUROD4 is upregulated before FOXO1 expression [NEUROD4 and FOXO1 and other co-factors involved in this transcriptomic regulatory network are largely unestablished.Among other genes, scRNA-seq shows upregulation of totrophs . Pituitaof GHRHR . Additiontiation . Howeverpression . The temCEBPD in somatotrophs suggests that differentiating pituitary cells actively repress altered cellular fates. In cell culture, overexpression of CEBPD results in the inhibition of PRL expression and lactotroph proliferation, whereas CEBPD knockdown using small interfering RNA leads to increased PRL expression [CEBPD to inhibit other PIT1 lineage differentiations. This is particularly of interest because the traditional model of pituitary cell differentiation generally views lineage-specific transcription factors as promotors of cellular fate, whereas inhibitory mechanism, such as the one seen with CEBPD, is not well recognized. Transcription factors may allow cellular differentiation bi-directionally promoting cell fate and repressing altered fate- which may explain why cell fates are so resistant to change.Interestingly, the overexpression of pression . Thus, iRecent studies suggest that somatotroph cells are not homogeneous but rather heterogenous in transcriptomic signature. SnRNA-seq of adult mice pituitary shows two distinct sub-clusters of somatotrophs . In the PRL requires the Ras-activation of PIT1 [POU4F1 and NR4A2 also increase PRL expression in PIT1 cells. NR4A2 directly binds to the PRL promotor, whereas POU4F1 enhances PRL expression without binding to the PRL promotor, possibly through unidentified regulatory elements [POU4F1 and NR4A2 results in more robust PRL expression than seen with POU4F1 alone, suggesting their synergistic effect on PRL [POU4F1 seems required for lactotroph differentiation, as NR4A2 without POU4F1 is insufficient to increase PRL.In normal lactotrophs, transcription of the hallmark gene  of PIT1 . POU4F1 elements . Co-exprt on PRL . NotablyZBTB20 in lactotrophs compared to somatotrophs and thyrotroph [ZBTB20 knock-down in vitro results in decreased PRL promotor activity, whereas ZBTB20 overexpression, in the presence of PIT1, results in increased PRL promotor activity [ZBTB20 knock-down mice, lactotroph development is inhibited at the early stage of somatolactotropic precursor cells [ZBTB20 regulates lactotroph specification, likely at the somatolactotropic stage, although the exact mechanism is still not well understood. In addition to its vital function in differentiating lactotrophs, ZBTB20 is critical for PRL expression in mature lactotrophs, at least in mice [In addition, scRNA-seq of the human fetal pituitary identifies upregulation of yrotroph . ZBTB20 activity . In ZBTBor cells . These s in mice .PRL, female lactotrophs, compared to male lactotrophs, express more genes involved in neurotransmission and endocrine release machinery, such as drd4 (dopamine receptor), gal , and grik1  [Single-cell transcriptomic analysis of adult mice and rat pituitaries points to significant sex-specific differences in gene expressions in lactotrophs. This is not surprising given that sexual maturation, reproduction, and development require sex-dependent endocrine demands and regulation. In addition to expressing a higher level of bunit 1) ,32. Whilbunit 1) , suggestGATA and PIT1 interact to drive TSHβ gene expression [GATA2 may involve two SoxC family genes, SOX4 and SOX11, which are expressed before GATA2 expression and bind to the regulatory region of GATA2 in thyrotroph precursor cells [GATA2 expression in thyrotroph cells and a large area of accessible chromatin upstream of GATA2, respectively [GATA2 knock-out mice show transient loss of thyrotroph cells and decreased production of TSH in response to hypothyroidism induction, in addition to compromised gonadotroph function [GATA2 knock-out mice indicates that GATA2 alone does not dictate thyrotroph cell fate and maintenance. In the same study, GATA2 knock-out mice exhibit elevated levels of other transcripts such as GATA3, pointing to a possible feedback regulation or compensation mechanism in thyrotroph specification. Given that some pituitary lineage-specific transcription factors seem dispensable rather than required for terminal cell fate, the pituitary transcription cascade may not be linear as traditionally modeled but rather a multi-directional network involving multiple transcription factors with subtle influence and innate compensatory mechanisms.In normal thyrotrophs, transcription factors pression ,35. ScRNor cells . Bulk RNfunction . HoweverISL1 is expressed in differentiating and postnatal thyrotroph cells [ISL1 transcripts in a mice model of thyrotroph hyperplasia (Cga-/-) [ISL1 in TSH-Cre mice results in a phenotype resembling hypothyroidism, it does not entirely ablate thyrotroph cells, similar to GATA2 knock-out mice [ISL1 may be more critical for cell functionality than cell differentiation, although delineating these differences may be difficult. Additionally, as previously mentioned, multiple transcription factors may simultaneously maintain normal homeostasis, and experimentally manipulating  one transcription factor at a time, while sufficient to decrease cellular function such as hormone secretion, may not be enough to ablate a pituitary lineage.The transcription factor ph cells . It is i(Cga-/-) . Howeverout mice . One expRXRG and DACH1 as two enriched genes in thyrotroph cells [RXRG and DACH1 are downregulated in the other two terminal PIT1 cell lines . Thus, RXRG and DACH1 may be specific for thyrotroph development and serve as potential transcriptomic markers to delineate thyrotroph from somatotroph and lactotroph. Whether targeted silencing of RXRG and DACH1 in differentiating thyrotroph cells is sufficient to reduce or revert its neuroendocrine cellular phenotype remains unanswered.ScRNA-seq on the human fetal pituitary identifies ph cells . They arSF1 , a zinc finger nuclear receptor that regulates multiple sex determination and reproduction genes in adrenal glands, gonads, and hypothalamus [SF1 partially depends on the binding of estrogen to estrogen-receptor-alpha , which mediates the chromatin remodeling of the SF1 enhancer and promotor in vitro [SF1 expression [SF1 knock-out in alpha-GSU-Cre mice results in hypogonadism and decreased expression of LH and FSH. Interestingly, SF1 may be important but not necessary for gonadotroph differentiation since a high dose of exogenous GnRH can rescue the loss of LH expression in SF1 knock-out mice [Differentiation of normal gonadotrophs involves thalamus ,38. The in vitro . ScRNA-spression . PituitaGATA2, expressed earlier than SF1, also plays a role in gonadotroph differentiation since GATA2 inactivation results in reduced gonadotrophin expression [FOXP2, a novel forkhead homeobox transcription factor, in the gonadotrophs [FOXP2 co-localizes with LH and FSH in vivo [pression . In addidotrophs . FOXP2 c in vivo , but itsGATA-positive/NR5A1-negative, “pre-gonadotroph” intermediate cell state [NR5A1-positive gonadotrophs characterized by different expression patterns of chorionic gonadotropin and FSH hormones. One of the subtypes is enriched with differentially expressed genes involved in the hormone biosynthesis process and exocytosis [MC2R, an ACTH receptor, suggesting that there may be crosstalk between gonadotroph and corticotroph lineage cells during development. Likewise, a subset of intermediate corticotrophs expresses some gonadotroph-related markers [In the human fetal pituitary, pseudotime analysis, which infers the ordering of cells in a lineage based on gene expression profiles of scRNA-seq, reveals two multi-step developmental trajectories that contain a ll state . The intocytosis . This suocytosis . Intrigu markers . WhetherThe pathogenesis of PitNETs is a complex process that involves abnormal transcriptomic changes, among other intrinsic and extrinsic drivers, that result in cell cycle dysregulation, loss of tumor suppressor factors, and signaling defects ,40. PitNUSP8 [USP8 mutations are specific to corticotroph PitNETs; other PitNETs do not display USP8 mutations on targeted sequencing [USP8 gain-of-function mutations result in the elevated deubiquitinating activity of EGFR and thereby increased accumulation of EGFR in the plasma membrane [POMC transcription and ACTH secretion in the mice corticotroph adenoma cell line  [POMC transcription in cultured cells derived from human corticotroph PitNETs [USP8-mutated corticotroph PitNETs show a higher incidence of EGFR expression, and USP8 knock-down reduces in vitro ACTH secretion [In 30–60% of ACTH-secreting PitNETs, dysregulation of ubiquitin-specific protease 8 (USP8) and the epidermal growth factor receptor (EGFR) pathway, neither of which are implicated in fetal corticotroph development, play a significant role ,43. WholUSP8 ,45,46. Uquencing . In normquencing ,48. USP8membrane ,45. Enha, AtT20) . In cont PitNETs . Consistecretion .POMC transcription and potentiate the stimulation effect of upstream cortisol-releasing hormone (CRH) [BRAF mutations have higher plasma ACTH and cortisol [In contrast, whole-exome sequencing studies in corticotroph PitNETs without USP8 mutations  identify BRAF and USP48 mutations. Similarly to USP8, BRAF and USP48 mutations are unique to corticotroph PitNETs. BRAF and USP48 mutations increase in vitro ne (CRH) ,51. In acortisol . While tNEUROD1 peaks at the intermediate states of corticotroph lineage development in the human fetal pituitary, whereas MNX1 remains upregulated in corticotroph cells in normal human adult pituitary. Bulk-RNA seq of corticotroph PitNETs shows that MNX1 is mildly upregulated, whereas NEUROD1 expression remains low [NEUROD1 [As previously mentioned, transcription factor ains low , suggest[NEUROD1 . These d[NEUROD1 , to thosCCND2) and zinc-finger protein 676 (ZNF676) and downregulation of death-associated protein kinase 1 (DAPK1) and TIMP metalloproteinase inhibitor 2 (TIMP2) in microarray analysis and qRT-PCR validation [CCND2 and DAPK1 regulate the cell cycle and TIMP2 regulates extracellular matrix homeostasis [ZNF676 plays a role in telomere homeostasis but the involvement of telomere dysregulation in PitNETs pathogenesis remains debated [SFRP2) in invasive corticotroph PitNETs [SFRP2 regulates Wnt signaling pathway and acts as a tumor suppressor [Corticotroph PitNETs exhibit heterogenous phenotypes, including variability in tumor aggressiveness and the ability to secrete ACTH. Transcriptomic analysis of corticotroph PitNETs suggests that tumor behavior correlates with differentially expressed genes . Comparelidation . CCND2 aeostasis ,57. ZNF6 debated ,59. In a PitNETs . SFRP2 rppressor ,61.PC1/3), signal peptidase (SPCS1), and dipeptidyl peptidase (DPP7)), secretory vesicle regulation ), and exocytosis regulation ) [PITX1, SIX3) which suggests a de-differentiated cellular state. In addition, silent corticotroph PitNETs exhibit characteristics of epithelial to mesenchymal transition  and mesenchymal matrix markers ), pointing to an increased tumor migration potential [Comparing silent and hypersecreting corticotroph PitNETs using scRNA-seq exhibits robust transcriptomic differences in their tumor cells. Compared to the hypersecreting corticotroph PitNETs, silent corticotroph PitNETs show significantly lower levels of genes involved in prohormonal processing ) . Silent otential ,63. Consotential . Thus, fPMAIP1, which encodes a pro-apoptotic noxa protein [PMAIP1, the hypersecreting corticotroph PitNETs evade apoptosis through proteasomal degradation of noxa protein. Consistently, proteasomal inhibitors  rescue the noxa protein and inhibit the growth of patient-derived corticotroph tumor cell lines [A recent transcriptomic study suggests proteasome-apoptosis pathway plays a role in corticotroph PitNET pathogenesis. ScRNA-seq of hypersecreting corticotroph PitNETs reveals upregulation of  protein . Interesll lines . While iGNAS, a gene that is part of signaling in normal fully developed somatotrophs but not in developing fetal somatotrophs, in up to 40% of somatotroph PitNETs. These mutations have been gain-of-function GNAS mutations resulting in constitutive activation of adenylyl cyclase and autonomous secretion of growth hormone [GNAS mutations are more commonly found in smaller-sized somatotroph PitNETs and are associated with the densely granulated variant on histology [GNAS mutation is associated with a better response to somatostatin receptor analogs but these findings are not consistent in the literature and require further investigation [GNAS-mutated somatotroph PitNETs are associated with higher expression of D2R, suggesting that dopamine agonists may benefit a subtype of acromegaly patients [SSTR5) is overexpressed in somatotroph PitNETs [Previous studies have identified somatic mutations in  hormone ,66,67,68istology . Some sttigation ,70,71,72patients . Another PitNETs , partial PitNETs . Somatos PitNETs ) that in PitNETs ,77,78. HNEUROD4 is upregulated in somatotroph PitNETs [NEUROD4 is a key transcription factor that works closely with FOXO1 in the terminal differentiation of normal somatotrophs, although whether NEUROD4 is upstream or downstream of FOXO1 is debated. Interestingly, despite NEUROD4 upregulation, there seem to be no significant changes in FOXO1 expression in somatotroph PitNETs [Similar to normal developing somatotroph cells,  PitNETs ,53. As p PitNETs . This suGHRHR, GH1, and GH2 in somatotroph PitNETs, consistent with their hyperfunctioning status [CG3, ANXA2, CLU, and GAA) and secretion  are upregulated. The same study identifies upregulation of AMIGO2 in somatotroph PitNETs, as well as in gonadotroph and lactotroph PitNETs. AMIGO2, a transmembrane protein, has been previously implicated in the pathogenesis of other cancers such as melanoma [A recent scRNA-seq study identifies high expressions of g status . Furthermelanoma  but its SF3B1) is a defining genetic signature [SF3B1 mutation does not appear in other types of PitNETs and is not implicated in fetal lactotroph development, although it is involved in diverse non-pituitary tumors [SF3B1 mutation shows increased estrogen-related receptor gamma (ESRRG) expression [SF3B1 mutant group has a higher affinity for PIT-1 and more robust transcriptional activation of PRL than canonical ESRRG, resulting in enhanced cell proliferation [SF3B1 mutation also downregulates Discs large 1 (DLG1), a tumor suppressor gene, in lactotroph PitNETs, thereby promoting tumor cell migration and invasion in vitro [In 20% of sporadic lactotroph PitNETs, a somatic mutation in splicing factor 3 subunit B1  show hyperprolactinemia and the development of lactotroph adenoma ,87, wher PitNETs ,90.αi), a downstream of D2R [In the subpopulation of lactotroph PitNETs resistant to dopamine agonist medications , studies suggest that there is a decreased expression of D2R isoform ,92 and im of D2R . Howeverm of D2R . Furtherm of D2R . NGFR rem of D2R ,97. Low Microarray transcriptomic studies have identified genes that are differentially expressed in non-invasive vs. aggressive-invasive lactotroph PitNETs. Non-invasive lactotroph PitNETs exhibit downregulation of genes implicated in proliferation  and upregulation of the gene  associated with tumor-suppressor DGL1 ,99. NotaGiven their rarity, representing ~1% of all PitNETs, the molecular mechanism of thyrotroph PitNET pathogenesis has not been well defined to date. Unlike corticotroph and somatotroph PitNETs, recurrent somatic mutations have yet to be identified in thyrotroph PitNETs . In studn = 8) in the gonadotroph PitNET (n = 29) cluster [Gonadotroph PitNETs  and null cell PitNETs are grouped together here because there remains some uncertainty about their classification and some overlap between them. While 2017 WHO classification of pituitary tumors differentiating silent gonadotroph tumors (SF1-positive) from null cell tumors (negative for cell type-specific transcription factors) is of clinical relevance given more invasiveness and worse clinical outcome in null cell adenomas ,109,110, cluster . ScRNA-s cluster . Thus, f cluster .As previously mentioned, there is evidence for crosstalk between gonadotroph and corticotroph lineage cells during normal fetal pituitary development . If suchLHB, ESR1, and GNRHR [CDKN1A, CDKN2A, ZFP36, BTG2, DLG5, and ZBTB16) and epithelial development . Thus, gonadotroph PitNETs may not just be hormonally silent but may represent a subtype of PitNETs with more globally diminished gene expression, which could explain why they overlap so much with null cell adenomas. Examining epigenetic regulation, especially silencing by DNA methylation, may provide more details on the mechanism of this diminished gene expression.The nonfunctional status of silent gonadotroph PitNETs is reflected by the downregulation of key genes involved in hormone production. For instance, compared to normal adult pituitary cells identified from PitNET resection, scRNA-seq of silent gonadotroph tumor cells exhibit downregulation of nd GNRHR . This cond GNRHR . In the MYO5A and IGFBP5 in invasive forms compared to non-invasive forms [MYO5A and IGFBP5 are involved in tumor cell migration and metastasis in other cancers [SPAG9, SKIL, MTDH, HOOK1, CNOT6L, and PRKACB) are particularly highly expressed in fast-growing tumors, as measured by tumor volume doubling time. While requiring further validations, these studies highlight the utility of molecular profiling in identifying potential predictive biomarkers for tumor growth potential.Transcriptomic profiling also identifies differential gene expression patterns in aggressive- and non-aggressive silent gonadotroph PitNETs. Microarray analysis of non-functional pituitary adenomas (comprising mostly gonadotroph PitNETs) shows higher expression of ve forms . MYO5A a cancers ,113,114.Recent advancements in transcriptomic biology, including the development of single-cell level RNA sequencing, have opened opportunities to molecularly dissect the pituitary, one of the most cellularly diverse regions of the brain. Deciphering the normal pituitary transcriptomic signatures gives us a better insight into the ontological origin and development of five types of endocrine cells, a process involving complex cascades of transcription factors that are still in the process of being established. In parallel with these observations about normal pituitary development, recent findings on PitNETs demonstrate both preservations and changes in transcription factor expression patterns. These studies of PitNETs also point to differentially expressed genes that drive various tumor behaviors, including hormone hypersecretion and tumor aggression. Understanding the comprehensive multiomic profiles of PitNETs will be essential in developing molecular profile-based therapies for PitNETs not curable with current treatment modalities and could eventually help align PitNETs with the breakthroughs being made in applying precision medicine to other tumors.


One of the major mysteries in science is how it is possible to pack the cellular chromatin with a total length of over 1 m, into a small sphere with a diameter of 5 mm “the nucleus”, and even more difficult to envisage how to make it functional. Although we know that compaction is achieved through the histones, however, the DNA needs to be accessible to the transcription machinery and this is allowed thanks to a variety of very complex epigenetic mechanisms. Either DNA (methylation) or post-translational modifications of histone proteins  play a crucial role in chromatin remodelling and consequently on gene expression. Recently the serotonylation and dopaminylation of the histone 3, catalyzed by the Transglutaminase type 2 (TG2), has been reported. These novel post-translational modifications catalyzed by a predominantly cytoplasmic enzyme opens a new avenue for future investigations on the enzyme function itself and for the possibility that other biological amines, substrate of TG2, can influence the genome regulation under peculiar cellular conditions. In this review we analyzed the nuclear TG2’s biology by discussing both its post-translational modification of various transcription factors and the implications of its epigenetic new face. Finally, we will focus on the potential impact of these events in human diseases. Several unique features, including its ubiquitous expression, widespread localization, binding to and hydrolysis of guanine nucleotides, distinguish TG2 from the other transglutaminases. TG2 changes from a closed to an opened form, under increased intracellular Ca2+ concentrations, exerting cross-linking and transamidase activities. Moreover, in addition to its cross−linking activity, TG2 might also act as a GTP−binding protein that mediates intracellular signalling by coupling the alpha−1 beta−adrenergic receptor to the phospholipase C−gamma1. Under physiological circumstances the enzyme may also act as protein disulphide isomerase (PDI) [2+ concentrations, it mainly acts as a GTPase-supporting growth. By contrast, when cells are injured and the intracellular Ca2+ concentration increases, it acts as a crosslinking, playing a key role in autophagy, or PDI enzyme and if the cellular damage is irreversible inducing cell death [Transglutaminases, also referred to as protein–glutamine g-glutamyltransferases EC 2.3.2.13), are a family of 8 active and 1 inactive (Band 3 of red blood cells) enzymes present in the human genome that catalyze the acyl-transfer reaction between glutamine and primary amines , 39. Tra, are a fse (PDI) . A serinse (PDI) , histonese (PDI)  and retise (PDI) . More rese (PDI) , 71, 73.se (PDI) . In the se (PDI) , 77. In ll death , 62. In ll death , 52, 75.ll death , 57.657LHMGLHKL664, near the C terminus, has been also identified [Although the first consistent report claiming the presence of TG2 in the nucleus was published more than 40 years ago , little entified . The nucentified , 85. In entified .Fig. 1WeIn the last 25 year several groups have shown that the nuclear TG2 can catalyse different post-translational modifications  of important transcription factors thus playing a direct key regulatory role on gene transcription  was the first tumor suppressor discovered and years of research have revealed that is a master regulator of biological pathways influencing cell fate, such as cell growth, cell-cycle, differentiation, senescence, self-renewal, replication, genomic stability and apoptosis. It has been shown that, the retinoblastoma gene product is post-translationally modified by a TG2-catalyzed reaction in the early stages of human leukemic monocyte lymphoma cells (U937 cells) undergoing apoptosis . In factSp1, a glutamine-rich protein belonging to the Sp-family of transcription factors, plays central roles in the regulation of a number of genes that influence cell survival and proliferation. Kojima and co-workers were the first who reported that in liver, consequently to the ethanol induced apoptosis, there is an enhanced nuclear accumulation of TG2 leading to the cross-linking inactivation of Sp1 followed by the reduced expression of the Sp1-dependent gene, c-Met. The comparison of the TG2 + / + vs mice TG2−/− mice showed markedly reduced hepatocyte apoptosis and Sp1 cross-linking following ethanol or anti-Fas treatment . A furthWe have recently demonstrated that TG2 regulates cellular proteostasis and cell's adaptation to stress by acting as a PDI . At the It has been recently shown that TG2 influences the overall cellular transcriptome profile and specifically the Wnt signaling. Indeed, TG2 is necessary for a proper induction of the Wnt pathway, that results strongly downregulated when the enzyme is absent . MechaniInterestingly, recent evidence showed a crosstalk between the Wnt and HSF1 pathways , 86. In Drosophila, transglutaminase (TG) crosslinks the nuclear factor-κB (NF-κB)-like transcription factor Relish active N-terminal fragment Relish-N to inhibit its nuclear translocation and the consequent regulation of many genes. It is interesting to note that the putative Relish-N TG-modified Gln residues are located in the DNA-binding region of Relish-N [About 20 years ago some reports highlighted the tight relationship existing between TG2 expression and NF-kB, the major regulator of genes involved in inflammatory and immune responses. Interestingly, both the genetic and the pharmacological inhibition of TG2 resulted in a reduced NF-kB/DNA interaction . An impoRelish-N . From thP53 is a multifunctional transcription factor that suppresses tumor growth through regulation of dozens of target genes with diverse biological functions. Its activity is inhibited in the vast majority of human tumors, either by gene mutations or overexpression of the p53 repressor MDM2.There is a general consensus that TG2 expression is upregulated in various tumors and this is associated with p53 instability , 42, 79.Interestingly it has also been reported that p53 is a substrate for a putative serine/threonine kinase activity of TG2 at p53 Ser(15) and Ser(20) residues that are critically important for the interaction of p53 with Mdm2 . HoweverIncreased TG2 promoted angiogenesis by inducing p53 degradation, leading to the activation of HIF-1α. Indeed, the TG2 dependent p53 depletion increased the availability of p300 for HIF-1α-p300 binding inducing angiogenesis .Interestingly, it has also been reported that ΔNp63α, another member of the p53 family, can be stabilized by TG2 leading to maintenance of cancer stem cell features .Post-translational modifications of histone are key epigenetic regulatory features that have important roles in the regulation of chromatin structure and in the activation and repression of gene transcription. The enzymes responsible for these epigenetic marks are often referred to as writers, erasers and readers. Writers introduce various chemical modifications on DNA and histones, erasers remove these chemical tags and readers identify and interpret those modifications . These hMany findings reported that TG2 is able to trigger different modifications of histones, making possible to suggest its role as a “writer”. More than two decades ago Shimizu et al. described the finding that an acyl transfer reaction, catalyzed by the nuclear TG2, could lead to the crosslinks of H2B and H4 histones in starfish sperm , 67. AccThese potential epigenetic features of the enzyme have been neglected for many years. However, recently Farrelly et al. convincingly demonstrated that the serotonin , an excitatory neurotransmitter, can be covalently attached to histone H3 by TG2. In this study it was shown that the glutamine 5 of the histone 3 (H3Q5) acts as the binding site for the serotonylation by TG2. The authors highlighted that H3Q5 serotonylation can occur in parallel with the trimethylation of the H3 on lysine 4 (H3K4me3), which has been shown to play an important role on gene transcription, by recruiting the TFIID and consequently the RNA polymerase II to the chromatin . In addiOther evidence about TG2 dependent modification of histones have been recently reported from the Maze’s group demonstrating that the TG2-catalyzed dopaminylation of histone 3 is associated with aberrant expression of addiction- and synaptic plasticity-related neuronal genes in response to cocaine consumption .A whole comparative transcriptomic analysis using TG2 knock out fibroblasts vs wild type confirmed a marked effect of TG2 on gene expression modulation . In partAnother unexpected emerging aspect about the chromatin/remodelling role of TG2 is that related to the regulation of the SWI/SNF (SWItch/Sucrose Non-Fermentable) complex. In fact, it has been shown that TG2 physically interacts with the BAF250a  protein which is a key component of the SWI/SNF complex (cBAF). In this case, TG2 catalyzes the covalent incorporation of polyamines into BAF250a . cBAF isWe have recently carried out the nuclear interactome of TG2 before and after doxorubicin treatment Fig. . Our datSo far, no mutations associated with TG2 have been described in mammals and extensive exome sequencing data have not uncovered individuals with homozygous loss-of-function variants for TGM2 gene . This seThe BAF complexes are generally considered as a transcriptional activator and antagonizes the Polycomb Repressor Complexes in the modulation of gene expression . NonetheTG2 has been shown to play a role in hepatocarcinogenesis and when absent there is a significant up-regulation of the TRIB3 mRNA expression . TRIB3 iIt has been shown that ARID1 A mutations are very frequent in ovarian cancers. They are mainly frame-shift or nonsense mutations determining loss of ARID1A expression and correlates with disease late-stages. Interestingly, BCL7A is also expressed at low levels in ovarian cancer tissues and is correlated with shorter survival status. Matei and co-workers have shown in a number of studies the important role played by TG2 in this tumor. In particular they have shown that TG2, by activating the NF-kB, inhibits apoptosis induced by cisplatin and modulates the epithelial-to-mesenchymal transition (EMT), thus contributing to increased ovarian cancer invasiveness and metastatic potentials . In linec [It has been very recently reported that the BCL7A-containing SWI/SNF/BAF complex regulates the Wnt signaling and the mitochondrial bioenergetics . Interesc , 64.The chromatin-remodelling BAF or SWI/SNF complexes have been reported to be involved in the induction of a subset of IFN-inducible genes . HoweverFinally, it has been shown that the hypoxia-inducible factor-1 alpha (HIF-1α) is directly regulated by the SWI/SNF chromatin-remodeling complex Indeed, the SWI/SNF components associate with the HIF-1α promoter resulting in a pronounced change in HIF-1α expression and consequently in its ability to transactivate target genes including TG2 , 64.It is becoming clear that the full understanding of the biological role of TG2 must encompasses its nuclear localization. The TG2 nuclear function can be exploited in many ways through its transamidating activity leading to the incorporation of primary amines into the histones as well as modifying transcriptional factors either by crosslinking or amines incorporation. The transamidating activity requires increased level of calcium which is released by the intracellular stores (ER mitochondria) mainly under stressful cellular conditions. However, TG2 can also acts as PDI as in the case of HSF1 trimerization or as scaffold protein without the calcium requirement. The demonstration of the post-translational incorporation of neurotransmitters in the H3K4me3 opens unexpected scenarios for the so far unclear comprehension of the involvement of TG2 in many biological processes, but more in general for the possibility that the enzyme can influence gene expression under peculiar cellular events leading to metabolic reprogramming. For instances the serotonylation of proteins has been shown to participate in several processes as neurotransmission, insulin secretion and vascular muscle contractility.TG2 catalyzed reactions may also lead to the covalent incorporation of other primary amines, such as putrescine, spermidine, spermine, histamine and noradrenaline. In this regard, it is well-documented the drastic increase of di- and polyamines observed in cancer cells . This meAn unexplored feature of TG2 biochemical activity, that can also play an important role in the chromatin post-translational modification, is its action as deamidating enzyme of specific glutamines. This TG2 activity plays a key role in celiac disease in which the deaminated gliadin acts as trigger for the production of pathogenetic antibodies specific for deamidated gluten peptides. Future studies should address as to whether the enzyme can also use this additional activity to modify the histones.Finally, it is important to define the nature of the interaction of TG2 with the BAF complex components that we have reported in this review. In particular, it is the protein acting as a scaffold under peculiar cellular circumstances or are its BAF interactors undergoing TG2-catalyzed posttranslational modifications? The elucubrations reported in this review led to a number of open questions that need to be addressed; however, it highlights the function of TG2 in the epigenetic field and suggests additional view in the regulation of chromatin post-translational modifications. The comprehension of these novel functions may significantly contribute to our understanding of TG2 complex function and to clarify its involvement in diseases.


Taurine, a sulfur-containing β-amino acid, is present at high concentrations in mammalian tissues and plays an important role in several essential biological processes. However, the genetic mechanisms involved in these physiological processes associated with taurine remain unclear. In this study, we investigated the regulatory mechanism underlying the taurine-induced transcriptional enhancement of the thioredoxin-interacting protein (TXNIP). The results showed that taurine significantly increased the luciferase activity of the human TXNIP promoter. Further, deletion analysis of the TXNIP promoter showed that taurine induced luciferase activity only in the TXNIP promoter region (+200 to +218). Furthermore, by employing a bioinformatic analysis using the TRANSFAC database, we focused on Tst-1 and Ets-1 as candidates involved in taurine-induced transcription and found that the mutation in the Ets-1 sequence did not enhance transcriptional activity by taurine. Additionally, chromatin immunoprecipitation assays indicated that the binding of Ets-1 to the TXNIP promoter region was enhanced by taurine. Taurine also increased the levels of phosphorylated Ets-1, indicating activation of Ets-1 pathway by taurine. Moreover, an ERK cascade inhibitor significantly suppressed the taurine-induced increase in TXNIP mRNA levels and transcriptional enhancement of TXNIP. These results suggest that taurine enhances TXNIP expression by activating transcription factor Ets-1 via the ERK cascade. Taurine (2-aminoethanesulfonic acid) is a free β-amino acid abundant in several tissues, such as muscles, the heart, brain, and eyes. Previous research indicates that taurine performs various functions, including as an antioxidant, osmoregulator, and bile acid conjugate, as well as in detoxification ,2. FurthTaurine is obtained directly from diet and is also synthesized endogenously from cysteine, which in turn is formed from methionine. Dietary taurine is absorbed by intestinal epithelial cells via the taurine transporter TAUT, SLC6A6). We previously reported that TAUT is regulated by various extracellular conditions, such as adaptive regulation and hyperosmolarity, as well as by tumor necrosis factor-α (TNF-α) ,6,7. FurA6. We prTXNIP suppresses its activity by interacting with thioredoxin ,11, and In the present study, we analyzed the transcription factors and signaling molecules involved in the taurine-induced increase in the transcriptional activity of TXNIP.Using reporter analysis, we previously reported that taurine increased the promoter activity of TXNIP . A reporWe also constructed a reporter vector containing the (−39/+256), (−39/+142), and (−39/+65) sequences of the promoter region of TXNIP. Taurine markedly increased reporter activity in the presence of a reporter vector containing the promoter region between (−39/+256), but did not increase activity in the case of (−39/+142) and (−39/+65) B. This rBased on the findings that the taurine response element is contained in the TXNIP promoter region between +143 and +256 , we nextThen, the TXNIP promoter region (+122/+256) was divided into three regions  and luciferase vectors each containing one region were constructed. Taurine significantly increased the promoter activity of TXNIP (+162/+218) , suggestFurther, the TXNIP promoter region (+162/+218) was divided into three regions , and a luciferase vector containing each region was constructed. Taurine significantly increased the promoter activity of TXNIP (+200/+218) , suggestThe promoter region of human TXNIP (+162/+218) was analyzed using a bioinformatics approach involving the TRANSFAC database. At the same time, deletion analysis of human TXNIP promoter was also performed. Then, the result of To confirm the enhancement of Ets-1 binding to the human TXNIP promoter region by taurine, a ChIP assay was performed. Caco-2 cells were cultured with 100 mM of taurine for 48 h and the cells were recovered. The lysate was immunoprecipitated using an anti-Ets-1 antibody. A real-time polymerase chain reaction (PCR) analysis was performed to determine whether the precipitated DNA contained the TXNIP promoter region with the Ets-1 response element. The results showed that taurine clearly increased Ets-1 binding to the TXNIP promoter region . We did We also examined whether taurine activates Ets-1 or not. Ets-1 is activated by Thr38 . Caco-2 To further elucidate the signaling pathway involved in taurine-induced TXNIP induction, we examined the effect of MAPK inhibitors on taurine TXNIP mRNA expression. The results showed that PD98059, an ERK1/2 pathway inhibitor, significantly suppressed the taurine-induced increase in TXNIP mRNA, whereas the p38 (SB203580) and JNK (SP600125) inhibitors did not .Next, to assess whether ERK1/2 was activated by taurine, the western blot analysis was performed. ERK1/2 was phosphorylated after 3 or 24 h of incubation with 100 mM of taurine . These rFinally, we examined the effect of PD98059 on the taurine-induced increase in TXNIP promoter activity (+200/+218). PD98059 significantly suppressed the taurine-induced increase in TXNIP promoter activity . This reIn the present study, we performed a detailed analysis of the mechanisms underlying the taurine-induced enhancement of TXNIP transcriptional activity. Our results demonstrate that taurine activates (phosphorylates) the transcription factor Ets-1 via the ERK signaling pathway and that the activated Ets-1 enhances the transcriptional activity of TXNIP by binding to the response sequence at +200/+218 in the TXNIP promoter region.A total of 100 mM of taurine increases the TXNIP promoter activity (+200/+218) about two-fold in The deletion analysis of the TXNIP promoter suggests that the response sequences of the transcription factors Tst-1 and Ets-1 in the 200/+218 region of the TXNIP promoter were the candidates for taurine response sequences. However, there have been no reports of promoter analyses on TXNIP in region 3′ from −39, and it is unknown what kind of transcription factor response sequences exist in this region. Therefore, we analyzed the sequences using JASPAR, a database of predicted transcription factor-binding sequences. These results revealed the presence of a putative Tst-1 transcription response sequence and an Ets-1 transcription factor response sequence in +210/+215 of the TXNIP promoter.Tst-1 belongs to a gene family with a Pit-Oct-Unc (POU) domain, and is reported to be expressed in the brain , and repMutation analysis suggests the involvement of Ets-1 in taurine-induced enhancement of TXNIP transcription. Subsequently, we examined whether taurine activates Ets-1 and found that taurine addition activated (phosphorylated) Ets-1 . ConsideThese findings suggest that taurine activates transcription factor Ets-1 via ERK and binds to the TXNIP promoter region, resulting in increased TXNIP transcriptional activity. The involvement of Ets-1 in the transcriptional activity of TXNIP has previously been reported in studies that observed induction of TXNIP by synthetic retinoids in osteosarcoma cells  and in tEts-1 is expressed in all tissues during embryonic development in mice and is reported to be crucial for morphogenesis . In thisA [Caco-2 cells are thought to recognize intracellular or extracellular taurine via unknown taurine receptors or binding proteins, leading to ERK-Ets-1 activation. In the brain, taurine binds to GABAA ,51,52 anA  receptorA  and are A . NoticeaA ; therefoA , suggestIn the present study, we revealed that taurine induced the transcriptional activity of TXNIP via the ERK and Ets-1 signaling pathways. These findings will hopefully contribute to the elucidation of the mechanisms underlying the diverse physiological effects of taurine and the discovery of new functions of taurine.The Caco-2 cell line, derived from human colon cancer tissue, was purchased from the American Type Culture Collection . Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Wako Pure Chemicals . Fetal bovine serum (FBS) was purchased from Sigma-Aldrich . Penicillin-streptomycin  and non-essential amino acids (NEAA) were purchased from Gibco . The lipofectamine reagent was purchased from Invitrogen , and the ExScript RT reagent kit and SYBR Premix Ex Taq for real-time PCR were obtained from Takara Bio . All other chemicals used were of reagent grade and commercially available.v/v), 1% NEAA (v/v), 100 U/mL of penicillin, and 100 µg/mL of streptomycin. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. The culture medium was replaced every other day. After reaching confluence, the cells were trypsinized with 0.1% trypsin and 0.02% EDTA in PBS and then subcultured. Caco-2 cells used in this study were between passages 35 and 79.Caco-2 cells were cultured in a medium consisting of DMEM, 10% FBS  was inserted into the pGL4-basic vector  as previously described . The humCaco-2 cells grown in a 24-well plate to 80% confluency were transiently transfected with 1 μg of the reporter vector and 0.05 μg of pRL-CMV using a lipofectamine reagent. Cells treated with or without 100 mM of taurine for 24 h were washed with PBS and lysed with Passive Lysis Buffer . Luciferase activity was measured using the dual-luciferase reporter assay  and an LB9507 Lumet luminometer .Total RNA was extracted from Caco-2 cells cultured with or without taurine using Isogen , according to the manufacturer’s instructions. Reverse transcription of the RNA was performed using the PrimeScript RT Reagent Kit , and first-strand cDNA was prepared from 0.5 μg of total RNA) and amplified using a SYBR Green Kit (TAKARA). The real-time PCR denaturation temperature was at 95 °C for 15 min, followed by 40 cycles of denaturation at 95 °C, at 60 °C for 15 s, and an extension at 72 °C for 10 s. The primer sequences were as follows: human TXNIP, 5′-ACGCTTCTTCTGGAAGACCA-3′ (forward), and 5′-AAGCTCAAAGCCGAACTTGT-3′ (reverse); β-actin, 5′-CCAGCACAATGAAGATCAAGA-3′ (forward) and 5′- AGAAAGGGTGTAACGCAACTAA-3′ (reverse). Real-time PCR was run on a LightCycler .Caco-2 cells were cultured in 6-well plates and incubated with taurine for 48 h. The cells were then homogenized, and the nuclear extract was prepared using the ChIP assay. The ChIP assay was performed using ChIP-IT Express , according to the manufacturer’s instructions. To quantify the number of DNA fragments containing the TXNIP promoter region bound by Ets-1 protein, real-time PCR was performed. The primer sequences were as follows: human TXNIP promoter, 5′-TCGGATCTTTCTCCAGCAAT-3′ (forward), and 5′-AAATCGAGGAAACCCCTTTG-3′ (reverse).3, 50 mM NaF, 20 mM β-glycerophosphate, 0.1% inhibitor cocktail . The cell homogenate was centrifuged at 20,000× g for 10 min at 4 °C. The supernatant was used for western blot analysis as described previously [Caco-2 cells were cultured in 6-well plates with or without taurine for several hours. The cells were collected with a cell scraper and suspended in lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM NaVOeviously . The prot-test, Dunnett’s test, or Tukey’s test.Data are expressed as mean ± SE. Statistical comparisons were performed using the Student’s