The role of RFX transcription factors in neurons and in the human brain

Abstract

RFX transcription factors (TFs) are conserved in animals, fungi and some amoebae, but not in algae, plants and protozoan species. The conservation is based on the protein sequence of the DNA binding domain (DBD). The RFX DBD recognizes and binds to a DNA sequence motif called the X-box. In addition to the DBD, most RFX TFs have a Dimerization domain (DIM). The DIM enables RFX TFs to form homo- or heterodimers in detecting the X-box motif, rendering the X-box often described as an imperfect palindromic sequence of two 6-bp half-sites with variable spacers.

So far, RFX TFs are known to regulate gene transcription in cell cycle, DNA repair, immune response, collagen transcription, insulin production, spermatogenesis and hearing. In animals, the most common feature of RFX TFs is their regulation of ciliogenesis and the maintenance of specialized functions of ciliated cells. Cilia are hair-like cell protrusions. They are present in all animals but absent in many species of fungi, amoebae and flowering plants. Based on the inner structure, cilia can be divided into two types, the primary cilia (one cilium per cell) and the motile cilia (either as mono-cilia or multiple-cilia per cell). The primary cilia are less understood despite being present on nearly every cell in the human body.

Humans have eight RFX genes (RFX1-8) which are expressed in diverse tissues and cell types. This thesis serves to expand knowledge of the RFX TF family in humans and their role in primary cilia and neurons, with interest in human brain development and function. We used databases (Paper I), human cell lines (Papers I and II) and the worm C. elegans (Paper III) as our materials for experimentation.

In Paper I, we performed an extensive survey of RFX1-8 expression by transcription start site (TSS) counts from the FANTOM5 database. RFX1-4 and RFX7 are prominently expressed in different brain tissues and spinal cord, making them the reference RFX TFs for neurons and the human brain. Furthermore, we predicted the regulation preference of RFX TFs based on co-clustering expression analysis with known RFX target genes. We also analyzed the positioning of the X-box motifs in the human genome and uncovered potential upstream regulators of RFX genes.

In Paper II, we explored the role of RFX TFs in the context of developmental dyslexia, a developmental disorder of the human brain. The dyslexia candidate genes DYX1C1, DCDC2 and KIAA0319 have functional X-box motifs in their promoter regions, as shown by luciferase reporter assay of wild-type versus mutated X-boxes. By siRNA knockdowns of RFX1-3, we showed a complex regulatory mechanism among RFX1-3 in regulating DYX1C1 and DCDC2. Additionally, both DYX1C1 and DCDC2 localize to the primary cilia.

In Paper III, we performed microarray analysis of target genes of DAF-19, the sole RFX TF of C. elegans, at three developmental stages (3-fold embryo, L1-larvae and adult). At all stages, DAF-19-regulated target genes were significantly enriched in neurons. Using transcriptional GFP reporter constructs, we observed that DAF-19-dependent target genes (both activated and repressed) affected only neurons, both ciliated and non-ciliated.

Altogether, we provided insight into the role of RFX TFs for primary cilia and neurons. We speculate that RFX TFs and primary cilia continue to play a defined role for mature neuron function in the human brain.