ChIPseq in Yeast Species: From Chromatin Immunoprecipitation to High-Throughput Sequencing and Bioinformatics Data Analyses

Lelandais, Gaëlle; Blugeon, Corinne; Merhej, Jawad

doi:10.1007/978-1-4939-3079-1_11

Cited by 8 publications

(11 citation statements)

References 24 publications

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“…Cross-linking of the cells and ChIP were performed as described previously 54 . The parental HTL (untagged strain) was grown and processed the same way to provide the mock-IP samples.…”

Section: Methodsmentioning

confidence: 99%

“…The parental HTL (untagged strain) was grown and processed the same way to provide the mock-IP samples. Sequencing of the IPs, Input DNAs and mock IPs samples and primary data analyses (quality controls and mapping of the reads) were performed as described previously 54 . Peak calling was performed with the bpeaks software 51 , using both the Input DNA and the mock IP as references.…”

Section: Methodsmentioning

confidence: 99%

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The CCAAT-Binding Complex Controls Respiratory Gene Expression and Iron Homeostasis in Candida Glabrata

Thiébaut

Delaveau

Benchouaia

et al. 2017

Sci Rep

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The CCAAT-binding complex (CBC) is a heterotrimeric transcription factor which is widely conserved in eukaryotes. In the model yeast S. cerevisiae, CBC positively controls the expression of respiratory pathway genes. This role involves interactions with the regulatory subunit Hap4. In many pathogenic fungi, CBC interacts with the HapX regulatory subunit to control iron homeostasis. HapX is a bZIP protein which only shares with Hap4 the Hap4Like domain (Hap4L) required for its interaction with CBC. Here, we show that CBC has a dual role in the pathogenic yeast C. glabrata. It is required, along with Hap4, for the constitutive expression of respiratory genes and it is also essential for the iron stress response, which is mediated by the Yap5 bZIP transcription factor. Interestingly, Yap5 contains a vestigial Hap4L domain. The mutagenesis of this domain severely reduced Yap5 binding to its targets and compromised its interaction with Hap5. Hence, Yap5, like HapX in other species, acts as a CBC regulatory subunit in the regulation of iron stress response. This work reveals new aspects of iron homeostasis in C. glabrata and of the evolution of the role of CBC and Hap4L-bZIP proteins in this process.

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“…Cross-linking of the cells and ChIP were performed as described previously 54 . The parental HTL (untagged strain) was grown and processed the same way to provide the mock-IP samples.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The CCAAT-Binding Complex Controls Respiratory Gene Expression and Iron Homeostasis in Candida Glabrata

Thiébaut

Delaveau

Benchouaia

et al. 2017

Sci Rep

Self Cite

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“…For ChIP, myc-tagged CgYAP strains were grown in YPD until exponential phase (OD = 0.8) and then stressing agents were added for 30 min. Cross-linking of the cells and ChIP were performed as described previously (Lelandais et al, 2016 ). The parental ΔHTU (untagged strain) was grown and processed the same way to provide the mock-IP samples.…”

Section: Methodsmentioning

confidence: 99%

“…The parental ΔHTU (untagged strain) was grown and processed the same way to provide the mock-IP samples. Sequencing of the IPs, Input DNAs and mock IPs samples and primary data analyses (quality controls and mapping of the reads) were performed as described previously (Lelandais et al, 2016 ). All experiments were performed twice and the reads of the replicate averaged before the peak calling step, except for CgYap5 for which one of the two replicates had poor read coverage and was not used for further analyses.…”

Section: Methodsmentioning

confidence: 99%

A Network of Paralogous Stress Response Transcription Factors in the Human Pathogen Candida glabrata

et al. 2016

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The yeast Candida glabrata has become the second cause of systemic candidemia in humans. However, relatively few genome-wide studies have been conducted in this organism and our knowledge of its transcriptional regulatory network is quite limited. In the present work, we combined genome-wide chromatin immunoprecipitation (ChIP-seq), transcriptome analyses, and DNA binding motif predictions to describe the regulatory interactions of the seven Yap (Yeast AP1) transcription factors of C. glabrata. We described a transcriptional network containing 255 regulatory interactions and 309 potential target genes. We predicted with high confidence the preferred DNA binding sites for 5 of the 7 CgYaps and showed a strong conservation of the Yap DNA binding properties between S. cerevisiae and C. glabrata. We provided reliable functional annotation for 3 of the 7 Yaps and identified for Yap1 and Yap5 a core regulon which is conserved in S. cerevisiae, C. glabrata, and C. albicans. We uncovered new roles for CgYap7 in the regulation of iron-sulfur cluster biogenesis, for CgYap1 in the regulation of heme biosynthesis and for CgYap5 in the repression of GRX4 in response to iron starvation. These transcription factors define an interconnected transcriptional network at the cross-roads between redox homeostasis, oxygen consumption, and iron metabolism.

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“…Immunoprecipitation was carried out by incubating the lysate with the monoclonal anti--FLAG M2-mouse primary antibody (Sigma Aldrich, F1804) and protein G Dynabead (Invitrogen, 10004D). Beads were washed sequentially in low salt, high salt and LiCl buffers, TE buffer and protein--DNA complex were eluted using elution buffer as reported earlier (Lelandais et al, 2016). Decrosslinking of the immuno--precipitated proteins were carried out by using a high concentration of NaCl and incubation at 65°C for 5 hours followed by proteinase--K treatment and DNA purification.…”

Section: Lysis and Chipmentioning

confidence: 99%

Genome-scale reconstruction of Gcn4/ATF4 networks driving a growth program

Srinivasan

Walvekar

Seshasayee

et al. 2020

Preprint

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How cells manage biomolecule supply to enable constructive metabolism for growth is a fundamental question. Methionine directly activates genomic programs where translation and metabolism are induced. In otherwise amino acid limitations, methionine induced ribosomal biogenesis, carbon metabolism, amino acid and nucleotide biosynthesis. Here, we find that the methionine--induced anabolic program is carbon--source independent, and requires the 'starvation-responsive' transcription factor Gcn4/ATF4. Integrating transcriptome and ChIP--Seq analysis, we decipher direct and indirect, methionine--dependent roles for Gcn4. Methionine--induced genes enabling metabolic precursor biosynthesis critically require Gcn4; contrastingly ribosomal genes are partly repressed by Gcn4. Gcn4 binds promoter--regions and transcribes a subset of metabolic genes, driving amino acid (particularly lysine and arginine) biosynthesis. This sustained lys/arg supply maintains high translation capacity, by allowing the translation of lys/arg enriched transcripts, notably the translation machinery itself. Thus, we discover how Gcn4 enabled metabolic--precursor supply bolsters protein synthesis demand, and drive a methionine--mediated anabolic program.

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ChIPseq in Yeast Species: From Chromatin Immunoprecipitation to High-Throughput Sequencing and Bioinformatics Data Analyses

Cited by 8 publications

References 24 publications

The CCAAT-Binding Complex Controls Respiratory Gene Expression and Iron Homeostasis in Candida Glabrata

The CCAAT-Binding Complex Controls Respiratory Gene Expression and Iron Homeostasis in Candida Glabrata

A Network of Paralogous Stress Response Transcription Factors in the Human Pathogen Candida glabrata

Genome-scale reconstruction of Gcn4/ATF4 networks driving a growth program

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