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RAB26 and RAB3D are direct transcriptional targets of MIST1 that regulate exocrine granule maturation.

ABSTRACT: Little is known about how differentiating cells reorganize their cellular structure to perform specialized physiological functions. MIST1, an evolutionarily conserved transcription factor, is required for the formation of large, specialized secretory vesicles in gastric zymogenic (chief) cells (ZCs) as they differentiate from their mucous neck cell progenitors. Here, we show that MIST1 binds to highly conserved CATATG E-boxes to directly activate transcription of 6 genes, including those encoding the small GTPases RAB26 and RAB3D. We next show that RAB26 and RAB3D expression is significantly downregulated in Mist1(-)(/)(-) ZCs, suggesting that MIST1 establishes large secretory granules by inducing RAB transcription. To test this hypothesis, we transfected human gastric cancer cell lines stably expressing MIST1 with red fluorescent protein (RFP)-tagged pepsinogen C, a key secretory product of ZCs. Those cells upregulate expression of RAB26 and RAB3D to form large secretory granules, whereas control, non-MIST1-expressing cells do not. Moreover, granule formation in MIST1-expressing cells requires RAB activity because treatment with a RAB prenylation inhibitor and transfection of dominant negative RAB26 abrogate granule formation. Together, our data establish the molecular process by which a transcription factor can directly induce fundamental cellular architecture changes by increasing transcription of specific cellular effectors that act to organize a unique subcellular compartment.

SUBMITTER: Tian X

PROVIDER: S-EPMC2820885 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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RAB26 and RAB3D are direct transcriptional targets of MIST1 that regulate exocrine granule maturation.

Tian Xiaolin X Jin Ramon U RU Bredemeyer Andrew J AJ Oates Edward J EJ Błazewska Katarzyna M KM McKenna Charles E CE Mills Jason C JC

Molecular and cellular biology 20091228 5

Little is known about how differentiating cells reorganize their cellular structure to perform specialized physiological functions. MIST1, an evolutionarily conserved transcription factor, is required for the formation of large, specialized secretory vesicles in gastric zymogenic (chief) cells (ZCs) as they differentiate from their mucous neck cell progenitors. Here, we show that MIST1 binds to highly conserved CATATG E-boxes to directly activate transcription of 6 genes, including those encodin ...[more]

PMID: 20038531

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Project description:UNLABELLED:Newborn neurons enter an extended maturation stage, during which they acquire excitability characteristics crucial for development of presynaptic and postsynaptic connectivity. In contrast to earlier specification programs, little is known about the regulatory mechanisms that control neuronal maturation. The Pet-1 ETS (E26 transformation-specific) factor is continuously expressed in serotonin (5-HT) neurons and initially acts in postmitotic precursors to control acquisition of 5-HT transmitter identity. Using a combination of RNA sequencing, electrophysiology, and conditional targeting approaches, we determined gene expression patterns in maturing flow-sorted 5-HT neurons and the temporal requirements for Pet-1 in shaping these patterns for functional maturation of mouse 5-HT neurons. We report a profound disruption of postmitotic expression trajectories in Pet-1(-/-) neurons, which prevented postnatal maturation of 5-HT neuron passive and active intrinsic membrane properties, G-protein signaling, and synaptic responses to glutamatergic, lysophosphatidic, and adrenergic agonists. Unexpectedly, conditional targeting revealed a postnatal stage-specific switch in Pet-1 targets from 5-HT synthesis genes to transmitter receptor genes required for afferent modulation of 5-HT neuron excitability. Five-HT1a autoreceptor expression depended transiently on Pet-1, thus revealing an early postnatal sensitive period for control of 5-HT excitability genes. Chromatin immunoprecipitation followed by sequencing revealed that Pet-1 regulates 5-HT neuron maturation through direct gene activation and repression. Moreover, Pet-1 directly regulates the 5-HT neuron maturation factor Engrailed 1, which suggests Pet-1 orchestrates maturation through secondary postmitotic regulatory factors. The early postnatal switch in Pet-1 targets uncovers a distinct neonatal stage-specific function for Pet-1, during which it promotes maturation of 5-HT neuron excitability. SIGNIFICANCE STATEMENT:The regulatory mechanisms that control functional maturation of neurons are poorly understood. We show that in addition to inducing brain serotonin (5-HT) synthesis and reuptake, the Pet-1 ETS (E26 transformation-specific) factor subsequently globally coordinates postmitotic expression trajectories of genes necessary for maturation of 5-HT neuron excitability. Further, Pet-1 switches its transcriptional targets as 5-HT neurons mature from 5-HT synthesis genes to G-protein-coupled receptors, which are necessary for afferent synaptic modulation of 5-HT neuron excitability. Our findings uncover gene-specific switching of downstream targets as a previously unrecognized regulatory strategy through which continuously expressed transcription factors control acquisition of neuronal identity at different stages of development.

Project description:Newborn neurons enter an extended maturation stage, during which they acquire excitability characteristics crucial for development of presynaptic and postsynaptic connectivity. In contrast to earlier specification programs, little is known aboutthe regulatory mechanisms that control neuronal maturation. The Pet-1 ETS (E26 transformation-specific) factor is continuously expressed in serotonin (5-HT) neurons and initially acts in postmitotic precursors to control acquisition of 5-HT transmitter identity. Using a combination of RNA sequencing, electrophysiology, and conditional targeting approaches, we determined gene expression patterns in maturing flow-sorted 5-HT neurons and the temporal requirements for Pet-1 in shaping these patterns for functional maturation of mouse 5-HT neurons. We report a profound disruption of postmitotic expression trajectories in Pet-1 / neurons, which prevented postnatal maturation of 5-HT neuron passive and active intrinsic membrane properties, G-protein signaling, and synaptic responses to glutamatergic, lysophosphatidic, and adrenergic agonists. Unexpectedly, conditional targeting revealed a postnatal stage-specific switch in Pet-1 targets from 5-HT synthesis genes to transmitter receptor genes required for afferent modulation of 5-HT neuron excitability. 5-HT1a autoreceptor expression depended transiently on Pet-1, thus revealing an early postnatal sensitive period for control of 5-HT excitability genes. Chromatin immunoprecipitation followed by sequencing revealed that Pet-1 regulates 5-HT neuron maturation through direct gene activation and repression. Moreover, Pet-1 directly regulates the 5-HT neuron maturation factor Engrailed 1, which suggests Pet-1 orchestrates maturationthrough secondary postmitotic regulatoryfactors. The early postnatal switch in Pet-1targets uncovers a distinct neonatal stage-specific function for Pet-1, during which it promotes maturation of 5-HT neuron excitability. 5-HT neuron mRNA profiles of E11.5, E15.5, and postnatal (P1-P3) wild type (WT) and Pet-1-/- mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. Myc-tagged Pet-1 ChIP-seq was performed on E12.5 to E14.5 hindbrains and sequencing using NextSeq 500.

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RAB26 and RAB3D are direct transcriptional targets of MIST1 that regulate exocrine granule maturation.

Publications

RAB26 and RAB3D are direct transcriptional targets of MIST1 that regulate exocrine granule maturation.

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