bHLH transcription factors in neural development and reprogramming

Question 1

Describe the structure of a BHLH protein.

Answer 1

HLH= helix loop helix (two alpha helices with non-conserved loop region used for dimerization
basic region used for DNA binding

Question 2

What is the bHLH consensus sequence?

Answer 2

E-box motif: CANNTG

Question 3

What is the difference between Class I and II bHLH factors?

Answer 3

Class I: ubiquitously expressed. Encoded by Tcfe2a, Tcf4, Tcf12.
Class II: tissue-specific expression profiles.

Question 4

What is the murine acahete-schute homolog in neural bHLH genes?

Answer 4

Ascl1/Mash1

Also Nscl family genes

Question 5

What are the atonal-related bHLH genes expressed in murine neural lineages?

Answer 5

Neurogenins, Neurod, Atonal, Olig families

Question 6

What is one difference between the function of proneural bHLH genes in Drosophila and in vertebrates?

Answer 6

In Drosophila, bHLH genes are expressed in uncommitted ectodermal cells to confer neural identity to these cells.
In vertebrates, proneural genes are expressed AFTER neural determination has occurred in cells that have already acquired a neural identity.
In both species, they are necessary and sufficient to promote neural differentiation and to specify neuronal subtype identities.

Question 7

Can proneural genes induce Notch signaling?

Answer 7

YES! Specifically the expression of Delta ligands like Dll1 and Dll3

Question 8

What are some of the gene families expressed at later stages in development (the differentiation bHLH genes) which are expressed in committed neuronal/glial precursors or in post mitotic neuronal or glial lineages?

Answer 8

NeuroD, Nscl, Olig families

Question 9

Describe 1) which cell pops N1 and N2 are expressed in, 2) their timelines of expression in neocortex.

Answer 9

Both expressed in cortical/pallial progenitors in VZ, lesser extent SVZ.
N1 expressed at onset of neurogenesis (E10), tapers around E15.5. N2 starts E10-E17 and beyond.

Question 10

Describe spatial distributions of N1 and N2 transcripts.

Answer 10

N1 more abundant in lateral domains.

N2 distributed throughout VZ.

Question 11

What is N1 N2 involvement in Cajal Retzius neuron formation?

Answer 11

N2 is required for a subset of these cells. Fewer CR cells in N2-/- brains. More CR cells in N1-/- brains. N1 limiting N2 function?

Question 12

What is N1N2 involvement in lateral piriform cortex formation?

Answer 12

BOTH are required together to generate lot1+ CR neurons. None in double mutant cortices.

Question 13

What is N1N2 involvement in subplot neuron formation?

Answer 13

N2 mutants have reduce and disorganized subplot neurons. In part this is because Ascl1 is unregulated, leading to misspecification of early-born neurons to aberrant GABAergic identity. N2/Ascl1 form genetic switch.

Question 14

What is the involvement of N1/N2 in the expression of markers conferring dorsal regional identity and glutamatergic phenotype in neocortex?

Answer 14

Both are required and sufficient to induce when misexpressed before E14.5.

N2 can induce NeuroD and Nscl1 families in cortical and subcortical progenitors.

Question 15

Which has a role in gliogenic repression: N1 or N2? How?

Answer 15

N1 (at least that’s all we know of so far). Repressed glial fate by 1) sequestering CBP-Smad-Stat3 transcriptional complexes away from regulatory regions of genes involved in astrogliogenesis, 2) activates expression of miR-9, an micro RNA that targets components of Jan-Stat pathway, including Lift-beta, Gp130, Jak1.

Question 16

Are N1 and N2 involved in neocortical neuron migration as well?

Answer 16

Yes. They are required for proper migration of neocortical neurons, especially deep layer neurons.
N2 residue Y241 regulates the polarity of the leading process during neuronal migration. Phosphorylation of this residue inhibits RhoA activity. N2 induces transcription of Rnd2, GTP-binding protein, which inhibits RhoA.

Question 17

Is there evidence that N2 regulates axonal projection patterns in the neocortex? Explain.

Answer 17

Yes. Thalamocortical axons (targeting subplot), corticofugal axons (project sub cortically from layers VI and V), and callosal axons (target midline) are all disrupted in N2 mutant cortices.

Question 18

What are some of the reasons for which individual bHLH factors can specify distinct cell fates in different domains?

Answer 18

• • regulated through post-transcriptional and post-translational events
• • cross-regulatory interactions (example: N2 required to repress Ascl1 expression in retina and forebrain). These repressive functions of proneural genes were not expected as they were considered to be transcriptional activators. Unravelling the regulatory interactions between bHLH genes and the extent to which they are redundant or have distinct functions, still remains a challenge

Question 19

What is the association between proneural bHLH factors and the Notch signaling pathway? Describe what happens when the Delta ligand interacts with a Notch receptor on a neighbour cell. What do Hes genes do?

Answer 19

Net effect: proneural bHLH factors transactivate Notch ligands (Dll1, Dll3), which then bind to Notch receptors on NEIGHBORING cells to repress proneural expression through ‘lateral inhibition’

Delta ligand binds Notch on another cell–> Notch is cleaved–> NICD translocates to nucleus where it interacts with Rbpj (CSL) to activate transcription of Hes genes. Hes genes then recruit Groucho/TLE co-repressor complexes to N-boxes (CACNAG) in promoter and enhancer elements to repress gene transcription. High Hes expression–> repression of proneural gene expression. Hes proteins can also bind and sequester E-proteins which would normally dimerize with proneural genes.

Question 20

Proneural activity in one cell may lead to proneural repression in neighbour cells. Is this repression stable?

Answer 20

No. Hes proteins are auto regulatory, so they can repress their own promoters, and can initiate oscillatory promoter activity. Within a given cell, Hes oscillations create a state whereby proneural genes and Hes genes oscillate inversely with each other in 2-3 hr cycles. Proneural genes thus create intercellular inhibition, while Hes genes create intracellular oscillations.

Question 21

How are N2 and N1 regulated by Wnt signaling?

Answer 21

Wnt can induce N2 expression and likely directly positively regulates N1 expression (there are Lef binding sites in N1).
Wnt signaling components are sufficient to induce ectopic expression of N1 and N2 in ventral telencephalon.
Wnt regulation of N2 is temporally restricted: Wnt disruption reduces N2 expression during early but not late cortical neurogenesis.

Question 22

How is N2 regulated by BMP signaling?

Answer 22

Bmp7, secreted from cortical hem, meninges, and choroid plexus, is required after E14.5 to maintain N2 expression in SVZ progenitors in the cortex.

Question 23

How does Fgf signaling affect selection of cell fate?

Answer 23

Fgf and its downstream effectors cause switch from N2 to Ascl1 expression. So Fgf pathway decides which proneural gene is expressed.

Question 24

How is N2 activity regulated by phosphorylation?

Answer 24

N2 has conserved SP and TP sites that are P by proline-directed serine-threonine kinases (Cdk1, GSK3, ERK). In Xenopus, has rheostat-like effect with each P reducing DNA binding more –> limits neurogenesis.

Question 25

In murine neocortex, describe regulation of N2 by phosphorylation after E14.5.

Answer 25

After E14.5, Wnt signaling decreases, which increases Gsk3 activity, and increases N2 phosphorylation on SP sites. When N2 gets P’d, changes choice of dimerization partner, preferentially forming N2-E47 heterodimers rather than N2-N2 homodimers. N2-E47 dimers have reduced ability to induce neuronal differentiation.
Therefore phosphorylation of proneural proteins may be a conserved mechanism to reduce proneural activity at different dev stages but also to direct neuronal subtype specification.

Question 26

Describe the epigenetic regulation of Neurog1.

Answer 26

Expression is repressed by Polycomb group (PcG) proteins at later stages of development to allow gliogenesis to initiate. Also, a 4kb enhancer region that controls N1 expression generates a non-coding RNA (utNeurog1) that is required for efficient N1 transcription.

Question 27

How might proneural genes activate their downstream targets in somatic cells in which the chromatin surrounding neuronal differentiation genes is closed?

Answer 27

N2 can do this by binding to histone acetyltransferases like CBP/p300. Can also interact with demethylases to remove repressive H3K9me2 marks and facilitate addition of open chromatin marks H3K27ac and H3K4me3.

Question 28

Is the direct mutation of bHLH genes the underlying cause of most cancers?

Answer 28

No. Their dysregulation has been observed.

Question 29

What two cancers do gliomas comprise?

Answer 29

Astrocytomas (glioblastoma multiforme) and oligodendrogliomas.

Question 30

What percentage of all malignant brain tumours do gliomas represent?

Answer 30

80%

Question 31

Which proneural gene is expressed in many gliomas? How does it influence tutor growth?

Answer 31

Ascl1. Influences tutor growth through Wnt pathway regulation (KD of Ascl1 decreases Wnt)

Question 32

Why might Ascl1 status be considered as a prognostic of drug responsiveness?

Answer 32

Because higher Ascl1 expression may make tutors more responsive to Notch inhibition, which allows for the latent proneural activity of Ascl1 to be activated, leading to cell cycle exit and neuronal differentiation.

Question 33

Is there a link between bHLH factors an neuropsychiatric disorders?

Answer 33

Yes, but fairly weak. Polymorphism within N1 regulatory region linked to language deficits. A region linked to schizophrenia, Chr 5q31, contains the NEUROG1 locus in humans. Within schizophrenia patients, those with the polymorphism have decreased verbal memory, language skills, and visuospatial abilities, and decreased cerebral grey matter volume.