Mesenchymal-to-epithelial transitions require tissue-specific interactions with distinct laminins

Mesenchymal-to-epithelial transition (MET) converts cells from migratory mesenchymal to polarized epithelial states. Despite its importance for both normal and pathological processes, very little is known about the regulation of MET in vivo. Here we exploit midgut morphogenesis in Drosophila melanogaster to investigate the mechanisms underlying MET. We show that down-regulation of the EMT transcription factor Serpent is required for MET, but not sufficient, as interactions with the surrounding mesoderm are also essential. We find that midgut MET relies on the secretion of specific laminins via the CopII secretory pathway from both mesoderm and midgut cells. We show that secretion of the laminin trimer containing the Wingblister α-subunit from the mesoderm is an upstream cue for midgut MET, leading to basal polarization of αPS1 integrin in midgut cells. Polarized αPS1 is required for the formation of a monolayered columnar epithelium and for the apical polarization of αPS3, Baz, and E-Cad. Secretion of a distinct LamininA-containing trimer from midgut cells is required to reinforce the localization of αPS1 basally, and αPS3 apically, for robust repolarization. Our data suggest that targeting these MET pathways, in conjunction with therapies preventing EMT, may present a two-pronged strategy toward blocking metastasis in cancer.

Download Full-text

Dynamic tracking and identification of tissue-specific secretory proteins in the circulation of live mice

10.21203/rs.3.rs-90987/v1 ◽

2020 ◽

Author(s):

Jae Myoung Suh ◽

Kwang-eun Kim ◽

Isaac Park ◽

Jeesoo Kim ◽

Myeong-Gyun Kang ◽

...

Keyword(s):

Blood Plasma ◽

Mouse Liver ◽

Secretory Pathway ◽

Secretory Protein ◽

Protein Labeling ◽

Secretory Proteins ◽

Tissue Specific ◽

Dynamic Tracking

Abstract Here we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which labels secretory pathway proteins as they transit through the ER-lumen to enable dynamic tracking of tissue-specific secreted proteomes in vivo. We expressed iSLET in the mouse liver and demonstrated efficient in situ labeling of the liver-specific secreted proteome which could be tracked and identified within circulating blood plasma. iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets.

Download Full-text

The Pu.1 Locus Is Differentially Regulated at the Level of Chromatin Structure and Noncoding Transcription by Alternate Mechanisms at Distinct Developmental Stages of Hematopoiesis

Molecular and Cellular Biology ◽

10.1128/mcb.00905-07 ◽

2007 ◽

Vol 27 (21) ◽

pp. 7425-7438 ◽

Cited By ~ 40

Author(s):

Maarten Hoogenkamp ◽

Hanna Krysinska ◽

Richard Ingram ◽

Gang Huang ◽

Rachael Barlow ◽

...

Keyword(s):

T Cells ◽

Transcription Factor ◽

Transcription Factors ◽

B Cells ◽

Regulatory Element ◽

Precursor Cells ◽

Hematopoietic Stem ◽

Tissue Specific ◽

Specific Regulation

ABSTRACT The Ets family transcription factor PU.1 is crucial for the regulation of hematopoietic development. Pu.1 is activated in hematopoietic stem cells and is expressed in mast cells, B cells, granulocytes, and macrophages but is switched off in T cells. Many of the transcription factors regulating Pu.1 have been identified, but little is known about how they organize Pu.1 chromatin in development. We analyzed the Pu.1 promoter and the upstream regulatory element (URE) using in vivo footprinting and chromatin immunoprecipitation assays. In B cells, Pu.1 was bound by a set of transcription factors different from that in myeloid cells and adopted alternative chromatin architectures. In T cells, Pu.1 chromatin at the URE was open and the same transcription factor binding sites were occupied as in B cells. The transcription factor RUNX1 was bound to the URE in precursor cells, but binding was down-regulated in maturing cells. In PU.1 knockout precursor cells, the Ets factor Fli-1 compensated for the lack of PU.1, and both proteins could occupy a subset of Pu.1 cis elements in PU.1-expressing cells. In addition, we identified novel URE-derived noncoding transcripts subject to tissue-specific regulation. Our results provide important insights into how overlapping, but different, sets of transcription factors program tissue-specific chromatin structures in the hematopoietic system.

Download Full-text

Expression of Drosophila melanogaster gene encoding transcription factor GAGA is tissue-specific and temperature-dependent

FEBS Letters ◽

10.1016/s0014-5793(97)01010-7 ◽

1997 ◽

Vol 414 (2) ◽

pp. 285-288 ◽

Cited By ~ 4

Keyword(s):

Transcription Factor ◽

Drosophila Melanogaster ◽

Temperature Dependent ◽

Gene Encoding ◽

Tissue Specific

Download Full-text

In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters

Scientific Reports ◽

10.1038/srep11842 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 30

Author(s):

Suzanne M. K. Buckley ◽

Juliette M. K. M. Delhove ◽

Dany P. Perocheau ◽

Rajvinder Karda ◽

Ahad A. Rahim ◽

...

Keyword(s):

Transcription Factor ◽

Tissue Specific ◽

Specific Transcription Factor ◽

Luciferase Reporters

Download Full-text

Characterization of Tissue-Specific HIF-1 and HIF-2 Regulatory Elements of the Erythropoietin Gene

Blood ◽

10.1182/blood.v112.11.4763.4763 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4763-4763

Author(s):

Donghoon Yoon ◽

Hyojin Kim ◽

Minyoung Jang ◽

Jihyun Song ◽

Gregory E Arnold ◽

...

Keyword(s):

Bone Marrow ◽

Specific Binding ◽

Regulatory Element ◽

Regulatory Elements ◽

Specific Gene ◽

Mrna Levels ◽

Specific Element ◽

Α Subunit ◽

Tissue Specific

Abstract Hypoxia regulates erythropoiesis and other essential processes via hypoxia-inducible transcription factors (HIFs). HIFs are heterodimers that consist of an α subunit (3 isotypes with significant homology; HIF-1α, HIF-2α, HIF-3α), and a common b-subunit; HIF-1 and HIF-2, in some instances exhibiting tissue- and gene-specific gene regulation. Erythropoietin (EPO) was the first identified HIF-1 target gene with the defined HIF-1 binding sequence. However, subsequent works suggested that HIF-2 also regulates EPO transcription and that there are other regulatory elements of EPO gene (i.e. Kidney Inducible Element KIE, Negative Regulatory Element NRE, and Negative Regulatory Liver specific Element NRLE). In silico analysis of the human EPO genome found two additional potential HIF-binding elements in the KIE and NRE regions. The comparative analysis of phylogenically conserved sequences of human, mouse, dog, and rat Epo genes further refined these mouse Epo gene HIF-binding elements as mKIE, mNRE1, mNRE2, and mNRLE2. We treated mice in hypoxia chamber (8% O2) and monitored changes of Epo mRNA levels in liver, kidney, brain, spleen, and bone marrow. All tested tissues increased Epo transcription during hypoxia. Bone marrow, spleen, kidney, and brain showed a peak of induction of Epo transcript at 3 hours of hypoxia treatment, while liver reached the highest level at 6 hours. Mice were sacrificed and organs were harvested, and in vivo chromatin immunoprecipitation (ChIPs) was performed with antibodies against HIF-1α and HIF- 2α and tissue-specific binding regions were defined. The results from these studies are summarized below. HIF-1 mKIE rnNRE mNRE2 mNRLE2 Norm Hyp Norm Hyp Norm Hyp Norm Hyp Liver − + − − + − ? ? Kidney − + − − + − + − Brain − + − − − + − + BM − + − − − − − + Splsen − + − − − − − + HIF-2 mKIE mNRE mNRE2 mNRLE2 Norm Hyp Norm Hyp Norm Hyp Norm Hyp “+” denotes presence and “-” absence of binding of HIF-1 and HIF-2, “?” – indicates inconclusive results. “Norm” - normoxia, “Hyp” - hypoxia. Liver − + − − − + − + Kidney + − − − + − ? ? Brain − − − − − − − + BM − − − − − − + − Spleen − + − − − − − + In conclusion, we demonstrate the differential hypoxia-induced binding of HIF-1 and HIF-2 at different HIF binding elements in the tissues known to express Epo. Further studies will be required to define the function of these HIF-1 and HIF-2 binding elements in tissue specific Epo expression and their role in health and disease.

Download Full-text

In Vivo Mutational Analysis of the DNA Binding Domain of the Tissue-specific Transcription Factor, Pit-1

Journal of Biological Chemistry ◽

10.1074/jbc.270.43.25520 ◽

1995 ◽

Vol 270 (43) ◽

pp. 25520-25525 ◽

Cited By ~ 16

Author(s):

Jie Liang ◽

Scott Moye-Rowley ◽

Richard A. Maurer

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Mutational Analysis ◽

Binding Domain ◽

Dna Binding Domain ◽

Tissue Specific ◽

Specific Transcription Factor

Download Full-text

Rapid Maturation of Glycoprotein Hormone Free α-Subunit (GPHα) and GPHαα Homodimers

Molecular Endocrinology ◽

10.1210/me.2007-0051 ◽

2007 ◽

Vol 21 (10) ◽

pp. 2551-2564 ◽

Cited By ~ 9

Author(s):

Jean-Michel Krause ◽

Peter Berger ◽

Jordi Roig ◽

Vinod Singh ◽

Wolfgang E. Merz

Keyword(s):

Hela Cells ◽

Secretory Pathway ◽

Cell Types ◽

Subunit Interaction ◽

Glycoprotein Hormone ◽

Α Subunit ◽

Self Association ◽

Choriocarcinoma Cells ◽

Loop 2

Abstract The dynamics of glycoprotein hormone α-subunit (GPHα) maturation and GPHαα homodimer formation were studied in presence (JEG-3 choriocarcinoma cells) and absence (HeLa cells) of hCGβ. In both cases, the major initially occurring GPHα variant in [35S]Met/Cys-labeled cells carried two N-glycans (Mr app = 22 kDa). Moreover, a mono-N-glycosylated in vivo association-incompetent GPHα variant (Mr app = 18 kDa) was observed. In JEG-3 cells the early 22-kDa GPHα either associated with hCGβ, or showed self-association to yield GPHαα homodimers, or was later converted into heavily glycosylated large free GPHα (Mr app = 24 kDa). Micro-preparative isolation of intracellular GPHαα homodimers of JEG-3 cells and their conversion by reduction revealed that they consisted of 22-kDa GPHα monomers and not of large free GPHα. In HeLa cells, the large free GPHα variant was not observed, whereas GPHαα homodimers were present. Intracellularly, early GPHαα homodimers (35 kDa) and late variants (JEG-3: 44 kDa, HeLa: 39 kDa) were found. Both cell types secreted 45 kDa GPHαα homodimers. Large free GPHα and GPHαα homodimers were more rapidly sialylated than hCG αβ-heterodimers indicating a sequestration mechanism in the secretory pathway. In GPHαα homo- as well as hCG αβ-heterodimers the subunit interaction site, located on loop 2 of GPHα (amino acids 33–42), became immunologically inaccessible indicating similar spatial orientation of GPHα in both types of dimers. The studies demonstrate the formation, in vivo dynamics of GPHαα homodimers, and the pathways of the cellular metabolism of variants of GPHα, monoglycosylated GPHα and large free GPHα.

Download Full-text

Tissue-Specific HIF-1 and HIF-2 Regulatory Elements and Their Roles in Erythropoietin Gene Expression

Blood ◽

10.1182/blood.v118.21.1303.1303 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1303-1303

Author(s):

Donghoon Yoon ◽

Jihyun Song ◽

Hyojin Kim ◽

Bumjun Kim ◽

Hyejin Shin ◽

...

Keyword(s):

Regulatory Elements ◽

Mouse Strains ◽

Renal Tubules ◽

Mrna Levels ◽

Specific Element ◽

Α Subunit ◽

Tissue Specific ◽

Hypoxic Induction ◽

Hypoxia Treatment

Abstract Abstract 1303 Studies of the regulation of erythropoiesis by hypoxic induction of erythropoietin (EPO) transcription led to the discovery of oligonucleotide binding motifs, hypoxia responsive elements (HREs) essential for hypoxic upregulation of EPO. HREs bind hypoxia-inducible transcription factors (HIFs). HIFs are heterodimers of 3 α subunit isotypes; HIF-1α, HIF-2α, and HIF-3α, and a common β-subunit. EPO is expressed in diverse tissues, some regulated mainly by HIF-2 and others by HIF-1. Tissue-specific regulatory elements (i.e. Kidney Inducible Element KIE, Negative Regulatory Element NRE, and Negative Regulatory Liver specific Element NRLE) of the EPO gene spanning several kilobases were reported. Our in silico analysis of the human EPO genome found two additional canonical HIF-binding elements in the KIE and NRE regions, and comparative analysis of phylogenically conserved sequences of Epo genes refined these HIF-binding elements from several kbs to a few hundred nucleotides and further delineated them as mKIE, mNRE1, mNRE2, and mNRLE2. To further define the structure and function of these elements in vivo, we exposed mice to 8% O2 hypoxia and monitored hypoxic induction of Epo mRNA levels in the liver, kidney, brain, and spleen. Spleen, kidney, and brain had a peak induction of the Epo transcript at 3 hours of hypoxia treatment, while liver showed peak induction at 6 hours. At the end of the hypoxia treatment, the mice were sacrificed and organs were harvested, and in vivo chromatin immunoprecipitation (ChIPs) was performed with antibodies against HIF-1α and HIF-2α. The results from these studies are summarized below. “+” denotes presence and “−” absence of binding of HIF-1 and HIF-2. “Norm” - normoxia, “Hyp” - hypoxia. To further define the role of these elements in tissue-specific Epo expression, we cloned and mutated these elements with reporter constructs, and generated transgenic mouse strains. The reporter gene was expressed in renal tubules, the red pulp area of the spleen, and liver hepatocytes. By creating a designed point mutation to disrupt the function of KIE, we suppressed the hypoxia response in the kidney, while mutations of NRE1 and NRE2 abolished reporter expression in the liver. Studies of HREs in the brain are ongoing. In conclusion, we have refined the previously reported HREs to a few hundred bases (particularly, KIE is a 6 nucleotide bearing motif) and now demonstrate tissue-specific differential hypoxia-induced binding of HIF-1 and HIF-2 and their functional roles in EPO induction. This provides a background to examine the molecular basis of these elements in patients with aberrant EPO expression. Disclosures: Elliott: Amgen, Inc: Employment.

Download Full-text

In vivo tissue-specific chromatin profiling in Drosophila melanogaster using GFP-tagged nuclei

Genetics ◽

10.1093/genetics/iyab079 ◽

2021 ◽

Author(s):

Juan Jauregui-Lozano ◽

Kimaya Bakhle ◽

Vikki M Weake

Keyword(s):

Drosophila Melanogaster ◽

Cell Types ◽

Chromatin Accessibility ◽

Chromatin State ◽

Specific Cell ◽

Tissue Specific ◽

Histone Marks ◽

Chromatin Profiling ◽

Photoreceptor Neurons

Abstract The chromatin landscape defines cellular identity in multicellular organisms with unique patterns of DNA accessibility and histone marks decorating the genome of each cell type. Thus, profiling the chromatin state of different cell types in an intact organism under disease or physiological conditions can provide insight into how chromatin regulates cell homeostasis in vivo. To overcome the many challenges associated with characterizing chromatin state in specific cell types, we developed an improved approach to isolate Drosophila melanogaster nuclei tagged with a GFPKASH protein. The perinuclear space-localized KASH domain anchors GFP to the outer nuclear membrane, and expression of UAS-GFPKASH can be controlled by tissue-specific Gal4 drivers. Using this protocol, we profiled chromatin accessibility using an improved version of Assay for Transposable Accessible Chromatin followed by sequencing (ATAC-seq), called Omni-ATAC. In addition, we examined the distribution of histone marks using Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and Cleavage Under Targets and Tagmentation (CUT&Tag) in adult photoreceptor neurons. We show that the chromatin landscape of photoreceptors reflects the transcriptional state of these cells, demonstrating the quality and reproducibility of our approach for profiling the transcriptome and epigenome of specific cell types in Drosophila.

Download Full-text