KLF6 and STAT3 co-occupy regulatory DNA and functionally synergize to promote axon growth in CNS neurons

Abstract

Members of the KLF family of transcription factors can exert both positive and negative effects on axon regeneration in the central nervous system, but the underlying mechanisms are unclear. KLF6 and −7 share nearly identical DNA binding domains and stand out as the only known growth-promoting family members. Here we confirm that similar to KLF7, expression of KLF6 declines during postnatal cortical development and that forced re-expression of KLF6 in corticospinal tract neurons of adult female mice enhances axon regeneration after cervical spinal injury. Unlike KLF7, however, these effects were achieved with wildtype KLF6, as opposed constitutively active mutants, thus simplifying the interpretation of mechanistic studies. To clarify the molecular basis of growth promotion, RNA sequencing identified 454 genes whose expression changed upon forced KLF6 expression in cortical neurons. Network analysis of these genes revealed sub-networks of downregulated genes that were highly enriched for synaptic functions, and sub-networks of upregulated genes with functions relevant to axon extension including cytoskeleton remodeling, lipid synthesis and transport, and bioenergetics. The promoter regions of KLF6-sensitive genes showed enrichment for the binding sequence of STAT3, a previously identified regeneration-associated gene. Notably, co-expression of constitutively active STAT3 along with KLF6 in cortical neurons produced synergistic increases in neurite length. Finally, genome-wide ATAC-seq footprinting detected frequent co-binding by the two factors in pro-growth gene networks, indicating co-occupancy as an underlying mechanism for the observed synergy. These findings advance understanding of KLF-stimulated axon growth and indicate functional synergy of KLF6 transcriptional effects with those of STAT3.SIGNIFICANCE STATEMENTThe failure of axon regeneration in the CNS limits recovery from damage and disease. These findings show the transcription factor KLF6 to be a potent promoter of axon growth after spinal injury, and more importantly clarify the underlying transcriptional changes. In addition, bioinformatics analysis predicted a functional interaction between KLF6 and a second transcription factor, STAT3, and genome-wide footprinting confirmed frequent co-occupancy. Co-expression of the two factors yielded synergistic elevation of neurite growth in primary neurons. These data point the way toward novel transcriptional interventions to promote CNS regeneration.

Co-occupancy analysis reveals novel transcriptional synergies for axon growth

“…Thus, analysis of TF co-occupancy can reveal novel instances of cooperation between TFs 21,22 . It was shown previously that the TF Klf6 is expressed in CST neurons during developmental periods of axon growth, is down-regulated during postnatal maturation, and that forced re-expression in adult neurons modestly enhances axon growth 7 . We therefore reasoned that during development, additional TFs likely co-occupy regulatory elements alongside Klf6 and cooperate to regulate transcription.…”

“…Using a well-established screening platform, postnatal cortical neurons received candidate genes by plasmid electroporation and were cultured at low density on laminin substrates, followed two days later by automated tracing to quantify neurite outgrowth. 7,12,24,28–31 . Nuclear-localized EGFP served to mark transfected neurons, and βIII tubulin immunohistochemistry labeled neuronal processes for automated tracing (Fig.2b) .…”

“…To date, progress has centered on identifying individual TFs whose ectopic expression in mature neurons leads to improved axon growth. For example, we showed previously that the TF Klf6 is developmentally downregulated and that viral re-expression drives improved axon growth in mature corticospinal neurons, which are critical mediators of fine movement 7 . The restoration of axon growth from this and other single-factor studies remains partial, however, indicating the need for additional intervention.…”

“…The restoration of axon growth from this and other single-factor studies remains partial, however, indicating the need for additional intervention. 7–12 . One likely possibility is that because multiple TFs generally act in a coordinated manner to regulate transcription, a more complete restoration of axon growth will depend on multiple TFs.…”

Krüppel‐like factor 6 promotes odontoblastic differentiation through regulating the expression of dentine sialophosphoprotein and dentine matrix protein 1 genes

“…In axon growth, KLF6 and STAT3 proteins may be affected by co‐occupancy of regulatory DNA as they have no mutual binding site that can be forecast in any interaction database (Wang et al . 2018). Osteogenic differentiation of mesenchymal stem cells was detected after STAT activation in monocytes (Nicolaidou et al .…”