Human Freud-2/CC2D1B: a novel repressor of postsynaptic serotonin-1A receptor expression.
BACKGROUND
Altered expression of serotonin-1A (5-HT1A) receptors, both presynaptic in the raphe nuclei and post-synaptic in limbic and cortical target areas, has been implicated in mood disorders such as major depression and anxiety. Within the 5-HT1A receptor gene, a powerful dual repressor element (DRE) is regulated by two protein complexes: Freud-1/CC2D1A and a second, unknown repressor. Here we identify human Freud-2/CC2D1B, a Freud-1 homologue, as the second repressor.


METHODS
Freud-2 distribution was examined with Northern and Western blot, reverse transcriptase polymerase chain reaction, and immunohistochemistry/immunofluorescence; Freud-2 function was examined by electrophoretic mobility shift, reporter assay, and Western blot.


RESULTS
Freud-2 RNA was widely distributed in brain and peripheral tissues. Freud-2 protein was enriched in the nuclear fraction of human prefrontal cortex and hippocampus but was weakly expressed in the dorsal raphe nucleus. Freud-2 immunostaining was co-localized with 5-HT1A receptors, neuronal and glial markers. In prefrontal cortex, Freud-2 was expressed at similar levels in control and depressed male subjects. Recombinant hFreud-2 protein bound specifically to 5' or 3' human DRE adjacent to the Freud-1 site. Human Freud-2 showed strong repressor activity at the human 5-HT1A or heterologous promoter in human HEK-293 5-HT1A-negative cells and neuronal SK-N-SH cells, a model of postsynaptic 5-HT1A receptor-positive cells. Furthermore, small interfering RNA knockdown of endogenous hFreud-2 expression de-repressed 5-HT1A promoter activity and increased levels of 5-HT1A receptor protein in SK-N-SH cells.


CONCLUSIONS
Human Freud-2 binds to the 5-HT1A DRE and represses the human 5-HT1A receptor gene to regulate its expression in non-serotonergic cells and neurons.
Hadjighassem
Mahmoud R
MR
Ottawa Health Research Institute (Neuroscience), University of Ottawa, Ottawa, Ontario K1H-8M5, Canada.
Austin
Mark C
MC
Szewczyk
Bernadeta
B
Daigle
Mireille
M
Stockmeier
Craig A
CA
Albert
Paul R
PR
D000328:Adult; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D003865:Major Depressive Disorder / Q000473:pathology; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D006801:Humans; D008297:Male; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009447:Neuroblastoma; D011180:Postmortem Changes; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism / Q000494:pharmacology; D044282:Receptor, Serotonin, 5-HT1A / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000502:physiology*; D014162:Transfection
C544137:CC2D1B protein, human; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D044282:Receptor, Serotonin, 5-HT1A