The Role And Mechanism Of Histone Methyltransferase SETD2 In Mice Cortical Development And Autistic-like Behaviors

The mammalian cerebral cortex is comprised of six-layered projection neurons,and is the most complex structure of nature.Tangentially,the cerebral cortex can be subdivided into distinct functional areas including the primary motor cortex(M1),visual cortex(V1),somatosensory cortex(S1),and auditory cortex(A1).The process of formation and specification of distinct functional areas within neocortex is called neocortical area patterning,also referred to arealization.Arealization defects is closely related to neurodevelopmental diseases.Autism spectrum disorder(ASD)is a neurodevelopmental disorder characterized by defects with social communication and by repetitive and/or restricted patterns of behaviors.Neuroanatomical and imaging studies indicated that 67%of children with ASD had enlarged frontal cortex areas.Sotos-like syndrome is an overgrowth syndrome with clinical characteristics of intellectual disability,speech delay,macrocephaly,facial dysmorphism,and ASD.SETD2,the lysine-36 of histone H3 tri-methyltransferase(H3K36me3),which loss-of-function mutations were found in some patients with ASD and Sotos-like syndrome.However,it remains elusive if the H3K36me3 methyltransferase activity of SETD2 is critical for brain development,social behaviors,and the pathogenesis of the Sotos-like syndrome.Since homozygous disruption of SETD2 in mice resulted in embryonic lethal at E10.5,we generated Setd2 conditional knockout mice(Setd2Emx1-cKO,Setd2Nestin-cKO,and Setd2Nex-cKO)using the Cre/loxP system.First,we found that Setd2Emx1-cKO mice had reduced exploration ability in the open filed test,social dysfunction in the three-chamber social test,decreased spatial memory and learning capacity in the Morris water maze experiment,and impaired motor learning ability in the rotarod test.Overall,loss of Setd2 in mice recapitulates some phenotypes including intellectual disability and defects in social communications found in Sotos-like and ASD patients.To explore the function of Setd2 in cortical area patterning,we analyzed the expression of area-specific genes by in situ hybridization(ISH)in postnatal day zero(P0)Setd2 conditional null mice.In Setd2Emx1-cKO,Setd2Nestin-cKO,and Setd2Nex-cKO mice cortex,the expression of Lmo4 and Epha7(motor and visual cortex specific markers)was medially retracted at the caudal motor cortex,and the expression of Ephrin-A5(a somatosensory cortex specific marker)was reduced.In P0 Setd2Emx1-cKO and Setd2Nestin-cKO mice cortex,the expression of Ror?(a somatosensory cortex specific marker)is medially extended at the cingulate cortex.In P7 Setd2Emx1-cKO mutants,Lmo4-labeled cells span the whole thickness of layer V of the somatosensory region,and Lmo4's expression in the frontal/motor cortices was greatly reduced.Similarly,the layered distribution of Cad8-expressing cells was also indiscernible in Setd2Emx1-cKO cortices,while Ror? expression was severely reduced in Setd2Emx1-cKO cortices.Serotonin(5-HT,a sensory cortex specific marker)immunohistochemical staining revealed that Setd2Emx1-cKO mice displayed prominent caudal expansion of the frontal/motor cortex,caudo-medial shift of the somatosensory cortex,and compromised antero-lateral barrel subfield(ALBSF).In Setd2Nex-cKO mice,the somatosensory cortex was caudo-medially shifted with compromised ALBSF.These results suggest that Setd2 is required for proper area patterning of the cerebral cortex.In control mice,motor and somatosensory cortex receives thalamocortical axons inputs from the ventral lateral nucleus(VL)and the ventral posterior nucleus(VP)respectively that terminate primarily in layer ?,and sends outputs from layer VI neurons to the same nucleus.However,the thalamocortical projections became obscure and dispersed in Setd2Emx1-cKO brains.The corticothalamic projections failed to project into VP and VL but showed overlapping characteristics in the internal capsule(IC)of Setd2Emx1-cKO brains.These data indicate that loss-of-function SETD2 in mice cortex disrupts the formation of cortico-thalamic-cortical circuits.RNA-seq analysis revealed that almost all clustered Protocadherins(cPcdh)family genes were significantly down-regulated in Setd2Emx1-cKO cortices.cPcdh heterozygous(Pcdh???+/-)mice showed similar area patterning defects as Setd2Emx1-cKO mice.Chromatin immunoprecipitation sequencing(ChIP-seq)detected nearly complete loss of H3K36me3 at cPcdh's enhancers.We also observed enhanced binding of DNMT3A and DNMT3B(especially DNMT3B)to some enhancers of cPcdh in Setd2Emx1-cKO cortices.Finally,we engineered the point mutated SETD2-expressing construct(SETD2L1815W,a point mutation identified in patients with Sotos-like syndrome)and transfected into SETD2-knockout 293T(293TSETD2-KO)cells.Results showed the Sotos-like syndrome-associated SETD2L1815W is devoid of tri-methyltransferase activity.In utero electroporation experiments showed that the H3K36me3 methyltransferase activity of SETD2 is required for the transcription of cPcdh.This is the first study that systematically revealed the role of SETD2 in cortical development,which not only gives direct evidence for understanding the function of epigenetic modification in area patterning,but also provides animal models and sheds light on molecular mechanism of the pathogenesis of Sotos-like syndrome and autism.